Sustained-release drug carrier composition

ABSTRACT

The present invention provides compositions for extended release of an active ingredient, comprising a lipid-saturated matrix formed from a biodegradable polymer. The present invention also provides methods of producing the matrix compositions and methods for using the matrix compositions to provide controlled release of an active ingredient in the body of a subject in need thereof.

FIELD OF THE INVENTION

The present invention provides compositions for extended release of anactive ingredient, comprising a lipid-based matrix with a biodegradablepolymer. The present invention also provides methods of producing thematrix compositions and methods for using the matrix compositions toprovide controlled release of an active ingredient in the body of asubject in need thereof.

BACKGROUND OF THE INVENTION

Lipid based drug delivery systems are well known in the art ofpharmaceutical science. Typically they are used to formulate drugshaving poor bioavailability or high toxicity or both. Among theprevalent dosage forms that have gained acceptance are many differenttypes of liposomes, including small unilamellar vesicles, multilamellarvesicles and many other types of liposomes; different types ofemulsions, including water in oil emulsions, oil in water emulsions,water-in-oil-in-water double emulsions, submicron emulsions,microemulsions; micelles and many other hydrophobic drug carriers. Thesetypes of lipid based delivery systems can be highly specialized topermit targeted drug delivery or decreased toxicity or increasedmetabolic stability and the like. Extended release in the range of days,weeks and more are not profiles commonly associated with lipid baseddrug delivery systems in vivo.

Ideally sustained release drug delivery systems should exhibit kineticand other characteristics readily controlled by the types and ratios ofthe specific excipients used. Advantageously the sustained release drugdelivery systems should provide solutions for hydrophilic, amphipathicas well as hydrophobic drugs.

Periodontitis

The use of systemic doxycycline and NSAIDs in combination therapy hasbeen shown to suppress tissue damage in the gingiva of chronicperiodontitis patients. Tissue damage is caused by the action ofpathogenic bacteria in combination with host matrix metalloproteinase(MMP) activity. Antibiotic treatment in combination withanti-inflammatory medication suppresses these two pathways. An increasein efficacy and reduction of side effects of treatment would be achievedby a means of releasing these medications locally in a controlledfashion.

Bone Augmentation

Bone diseases requiring bone augmentation include benign and malignantbone tumors, cancers situated in bones, infectious bone diseases, andother bone diseases of etiology related to endocrinology, autoimmunity,poor nutrition, genetic factors, and an imbalance between bone growthand resorption. Examples are diseases such as osteosarcoma/malignantfibrous histiocytoma of bone (PDQ), osteosarcoma, chondrosarcoma,Ewing's sarcoma, malignant fibrous histiocytoma, fibrosarcoma andmalignant fibrous histiocytoma, giant cell tumor of bone, chordoma,lymphoma, multiple myeloma, osteoarthritis, Paget's disease of bone,arthritis, degenerative changes, osteoporosis, osteogenesis imperfecta,bone spurs, renal osteodystrophy, hyperparathyroidism, osteomyelitis,enchondroma, osteochondroma, osteopetrosis, bone and joint problemsassociated with diabetes.

Immediate and delayed infection is a major complication in the field oforthopedics. Reducing the complications after orthopedic treatment willinduce the efficiency and success of the orthopedic treatment and insome cases it will reduce the mortality. There is also a need to allowtreatment in infected sites and to induce the efficacy of the treatmentin the infected sites.

Another important aspect in the field of orthopedics or orthopedicsurgery is the need to accelerate soft and hard tissue recovery inreparative and regenerative procedures.

Liposomes and Biodegradable Polymers in Drug Delivery

To date the use of lipids in conjunction with biopolymers has beencontemplated but these have not yet been introduced successfully intoclinical practice.

U.S. Pat. No. 3,773,919 to Boswell et al describes the use of polymersderived from alpha-hydroxycarboxylic acids, including lactic acid,glycolic acid, and copolymers thereof, and their use in sustainedrelease formulations. Such polymer exhibit slow biodegradability buttypically have limited drug-holding capacity.

Liposomes are described in U.S. Pat. No. 4,522,803 to Lenk et al.Liposomes typically exhibit adequate drug delivery drug-holding capacitybut relatively limited in vivo half-lives. Many different types ofliposomes have been developed for particular applications. Examples canbe found in U.S. Pat. Nos. 5,043,166; 5,316,771; 5,919,480; 6,156,337;6,162,462; 6,787,132; 7,160,554, among others.

U.S. Pat. Nos. 6,333,021 and 6,403,057 to Schneider et al disclosemicrocapsules having a biodegradable membrane encapsulating a gas core.The membrane, comprising water insoluble lipids with up to 75% by weightof biodegradable polymers, encapsulating a core filled with air or agas. The microcapsules may be non-coalescent, dry and instantlydispersible, and useful as delivery vehicles for therapeutically activeagents and/or as contrast agents for imaging of body organs. Themicrocapsules are produced by a method in which a water-in-oil emulsionis made from an organic solution comprising a dissolved lipid and anaqueous solution containing a surfactant. The freeze-dried mixture isre-dispersed in an aqueous carrier, and the microcapsules are dried. Thepresence of water throughout the process precludes formation of awater-resistant, lipid-saturated matrix; thus, these materials aresubject to bulk-type degradation in vivo.

U.S. Pat. Nos. 6,277,413 and 6,793,938 to Sankaram disclosebiodegradable lipid/polymer-containing compositions, formed by thefollowing process: a) forming a water-in-oil emulsion from a firstaqueous phase and a volatile organic solvent phase comprising a volatileorganic solvent, a biodegradable polymer or copolymer that is soluble inorganic solvent, and a lipid; b) dispersing the “water-in-oil” emulsioninto a surfactant-free second aqueous phase to form solvent spherules,and c) removing the volatile organic solvent from the solvent spherulesto form a microsphere composition suspended in the second aqueous phase.The methods disclosed utilize aqueous solutions, precluding formation ofa water-resistant, lipid-saturated matrix.

U.S. Pat. No. 4,882,167 to Jang discloses a controlled release matrixfor tablets or implants of biologically active agents produced by drydirect compression of a hydrophobic carbohydrate polymer, e.g. ethylcellulose; and a difficult-to-digest soluble component, i.e. a wax, e.g.carnauba wax, a fatty acid material, or a neutral lipid. Thecarbohydrate polymers utilized, are unsuitable for release on a scale ofweeks or months following administration by injection or implantation.In addition, the compositions are produced without any solvents (aqueousor organic), precluding formation of the homogenous lipid-saturatedmatrix structures.

US patent application 2006/0189911 to Fukuhira et al discloses ananti-adhesion membrane of a honeycomb film made of polylactic acid as abiodegradable polymer and a phospholipid. No disclosure is provided formodification of the membrane for use as a delivery system, e.g. forantibiotics or NSAID drugs. In addition, the disclosed membranes arerequired to be cast under conditions of high humidity, thus precludingformation of a water-resistant, lipid-saturated matrix; these implantsare accordingly subject to bulk-type degradation in vivo.

US patent application 2006/0073203 to Ljusberg-Wahren et al discloses anorally administrable composition comprising a dry mixture of polymer,lipid and bioactive agent, intended upon contact with water orgastrointestinal fluids to form particles comprising the lipid, thebioactive agent, and optionally also water. The polymers utilized,disintegrate in the digestive tract during the digestive process; e.g. atime period of less than one day. Such compositions are completelyunsuitable for release on a scale of weeks or months followingadministration by injection or implantation.

None of the prior art provides compositions adapted to achieve sustainedrelease or programmed release or controlled release from alipid-saturated polymeric matrix for periodontal or orthopedic uses.None of the above references demonstrates use of the disclosedcompositions in delivery of an NSAID compound, an antibiotic compound,or a compound useful for bone augmentation.

SUMMARY OF THE INVENTION

The present invention provides compositions for extended release of anactive ingredient, comprising a lipid-based matrix comprising abiodegradable polymer. The present invention also provides methods ofproducing the matrix compositions and methods for using the matrixcompositions to provide controlled release of an active ingredient inthe body of a subject in need thereof.

In one aspect, the present invention provides a matrix compositioncomprising: (a) a biodegradable pharmaceutically acceptable polymer inassociation with a first lipid having a polar group; (b) a second lipidselected from phospholipids having hydrocarbon chains of at least 14carbons; and (c) a pharmaceutical active agent, where the matrixcomposition is adapted for providing sustained release of thepharmaceutical agent. In specific embodiments, the polymer and thephospholipids form a matrix composition that is substantially free ofwater.

According to particular embodiments the biodegradable polymer comprisesa polyester selected from the group consisting of PLA (polylactic acid),PGA (poly glycolic acid), PLGA (poly (lactic-co-glycolic acid)) andcombinations thereof.

According to particular embodiments the first lipid having a polar groupis selected from a sterol, a tocopherol and a phosphatidylethanolamine.According to particular embodiments the first lipid is mixed with thebiodegradable polymer to form a non-covalent association.

According to some embodiments the second lipid comprises aphosphatidylcholine. According to some embodiments the second lipidcomprises a mixture of phosphatidylcholines. According to someembodiments the second lipid comprises a mixture of aphosphatidylcholine and a phosphatidylethanolamine, or any other typesof phospholipids.

Any type of drug molecule may be incorporated into the matrixcompositions for sustained and/or controlled release. According toparticular embodiments the pharmaceutically active agent is selectedfrom the group consisting of an antibiotic, an antifungal, an NSAID, asteroid, an anti-cancer agent, an osteogenic factor and a boneresorption inhibitor. According to alternative embodiments thepharmaceutical active agent is selected from a hydrophobic agent, anamphipathic agent or a water soluble agent. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the phospholipid is a phosphatidylcholine havingfatty acid moieties of at least 14 carbons. In another embodiment, thecomposition further comprises a phosphatidylethanolamine having fattyacid moieties of at least 14 carbons. In another embodiment, thecomposition further comprises cholesterol. In another embodiment, thematrix composition is homogeneous. In another embodiment, the matrixcomposition is in the form of a lipid-based matrix whose shape andboundaries are determined by the biodegradable polymer. In anotherembodiment, the matrix composition is in the form of an implant.

In some embodiments, the pharmaceutical active agent is an antibioticincorporated into the matrix composition. In some embodiments, theantibiotic has low water solubility. In another embodiment, theantibiotic is a hydrophobic antibiotic. In another embodiment, theantibiotic is an amphipathic antibiotic. In another embodiment, thecomposition further comprises a non-steroidal anti-inflammatory drug(NSAID). In another embodiment, the NSAID as well is incorporated intothe matrix composition. In another embodiment, the NSAID has low watersolubility. Each possibility represents a separate embodiment of thepresent invention.

In a particular embodiment, the present invention provides a matrixcomposition comprising: (a) biodegradable polyester; (b) a sterol; (c) aphosphatidylethanolamine having fatty acid moieties of at least 14carbons; (d) a phosphatidylcholine having fatty acid moieties of atleast 14 carbons; and (e) an antibiotic or antifungal agent. In anotherembodiment, the matrix composition comprises at least 50% lipid byweight. In another embodiment, the matrix composition is homogeneous. Inanother embodiment, the matrix composition is in the form of alipid-based matrix whose shape and boundaries are determined by thebiodegradable polymer. In another embodiment, the matrix composition isin the form of an implant.

According to alternative embodiments the antibiotic or antifungal agentis selected from a hydrophobic agent, an amphipathic agent or a watersoluble agent. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a matrixcomposition comprising: (a) biodegradable polyester; (b) a sterol; (c) aphosphatidylethanolamine having fatty acid moieties of at least 14carbons; (d) a phosphatidylcholine having fatty acid moieties of atleast 14 carbons; and (e) a non-steroidal anti-inflammatory drug(NSAID). In another embodiment, the matrix composition comprises atleast 50% lipid. In another embodiment, the NSAID has low watersolubility. In another embodiment, the NSAID is a hydrophobic NSAID. Inanother embodiment, the NSAID is an amphipathic NSAID. In anotherembodiment, the matrix composition is in the form of a lipid-basedmatrix whose shape and boundaries are determined by the biodegradablepolymer. In another embodiment, the matrix composition is in the form ofan implant. In another embodiment, the matrix composition ishomogeneous. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a matrixcomposition comprising: (a) biodegradable polyester; (b) a sterol; (c) aphosphatidylethanolamine having fatty acid moieties of at least 14carbons; (d) a phosphatidylcholine having fatty acid moieties of atleast 14 carbons; and (e) an osteogenic factor or a bone resorptioninhibitor. In another embodiment, the matrix composition comprises atleast 50% lipid. In another embodiment, the bone resorption inhibitorhas low water solubility. In another embodiment, the bone resorptioninhibitor is a hydrophobic bone resorption inhibitor. In anotherembodiment, the bone resorption inhibitor is an amphipathic boneresorption inhibitor. In another embodiment, the composition furthercomprises an NSAID. In another embodiment, the NSAID as well isincorporated into the matrix composition. In another embodiment, thematrix composition is in the form of a lipid-based matrix whose shapeand boundaries are determined by the biodegradable polymer. In anotherembodiment, the matrix composition is in the form of an implant. Inanother embodiment, the matrix composition is homogeneous. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a matrixcomposition comprising: (a) biodegradable polyester; (b) a sterol; (c) aphosphatidylethanolamine having saturated fatty acid moieties of atleast 14 carbons; (d) a phosphatidylcholine having saturated fatty acidmoieties of at least 14 carbons; (e) an active agent; and (f) atargeting moiety capable of interacting with a surface molecule of atarget cell. In another embodiment, the active agent is selected fromthe group consisting of an NSAID, an antibiotic, an antifungal agent, asteroid, an anti-cancer agent, an osteogenic factor and a boneresorption inhibitor. In another embodiment, the polymer and thephospholipid form a matrix composition that is substantially free ofwater. In another embodiment, the matrix composition is capable of beingdegraded in vivo to vesicles into which some or all the mass of thereleased active agent is integrated. In another embodiment, the matrixcomposition is capable of being degraded in vivo to form vesicles intowhich the active agent and the targeting moiety are integrated. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising a matrix composition of the present invention anda pharmaceutically acceptable excipient. In another embodiment, thematrix composition is in the form of microspheres. In anotherembodiment, the present invention provides a pharmaceutical compositioncomprising microspheres of the present invention and a pharmaceuticallyacceptable excipient. In another embodiment, the pharmaceuticalcomposition is in a parenterally injectable form. In another embodiment,the pharmaceutical composition is in an infusible form. In anotherembodiment, the excipient is compatible for injection. In anotherembodiment, the excipient is compatible for infusion. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a matrix composition of the present invention isin the form of an implant, following evaporation of the organicsolvents. In another embodiment, the implant is homogeneous. Eachpossibility represents a separate embodiment of the present invention.

In some embodiments, the biodegradable polyester of the presentinvention is associated with the sterol via non-covalent bonds. In someembodiments, the biodegradable polyester of the present invention isassociated with the sterol via hydrogen bonds.

In another embodiment, the process of creating an implant from acomposition of the present invention comprises the steps of (a) creatinga matrix composition according to a method of the present invention inthe form of a bulk material; and (b) transferring the bulk material intoa mold or solid receptacle of a desired shaped.

Also provided herein are methods for making the compositions of theinvention and methods of use thereof.

According to another aspect a matrix composition for sustained releaseof a pharmaceutical agent is generated by a process comprising:providing a first solution or dispersion of a volatile organic solventcomprising a biodegradable polymer and a first lipid having a polargroup; providing a second solution or dispersion comprising a secondvolatile organic solvent and a second lipid, the second lipid comprisingat least one phospholipid, and a pharmaceutical active agent; mixing thefirst and second solutions to form a homogeneous mixture; evaporatingthe volatile solvents to produce a homogeneous polymer phospholipidmatrix comprising a pharmaceutical active agent. The selection of thespecific solvents is made according to the specific drug and othersubstances used in the particular formulation intended to entrap aspecific active and to release it in a specific pre-planned rate andduration. The evaporation is conducted at controlled temperaturedetermined according to the properties of the solution obtained.

According to the present disclosure the use of different types ofvolatile organic solutions, and the absence of water throughout theprocess, enable the formation of homogeneous water-resistant, lipidbased matrix compositions. According to various embodiments the firstand second solvents can be the same or different. According to someembodiments one solvent can be non-polar and the other preferablywater-miscible.

In another embodiment, the matrix composition of methods andcompositions of the present invention is substantially free of water.“Substantially free of water” refers, in another embodiment, to acomposition containing less than 1% water by weight. In anotherembodiment, the term refers to a composition containing less than 0.8%water by weight. In another embodiment, the term refers to a compositioncontaining less than 0.6% water by weight. In another embodiment, theterm refers to a composition containing less than 0.4% water by weight.In another embodiment, the term refers to a composition containing lessthan 0.2% water by weight. In another embodiment, the term refers to theabsence of amounts of water that affect the water-resistant propertiesof the composition.

In another embodiment, the term refers to a composition manufacturedwithout the use of any aqueous solvents. In another embodiment,producing the composition using a process substantially free of water,as described herein, enables lipid saturation. Lipid saturation confersupon the matrix composition ability to resist bulk degradation in vivo;thus, the matrix composition exhibits the ability to mediate extendedrelease on a scale of several days, weeks or months.

In another embodiment, the matrix composition is essentially free ofwater. “Essentially free” refers to a composition comprising less than0.1% water by weight. In another embodiment, the term refers to acomposition comprising less than 0.08% water by weight. In anotherembodiment, the term refers to a composition comprising less than 0.06%water by weight. In another embodiment, the term refers to a compositioncomprising less than 0.04% water by weight. In another embodiment, theterm refers to a composition comprising less than 0.02% water by weight.In another embodiment, the term refers to a composition comprising lessthan 0.01% water by weight. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the matrix composition is free of water. Inanother embodiment, the term refers to a composition not containingdetectable amounts of water. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method ofproducing a matrix composition, the method comprising the steps of (a)combining with a non-polar, volatile organic solvent: (i) abiodegradable polyester and (ii) a sterol; (b) combining with awater-miscible, volatile organic solvent: (i) an active agent selectedfrom the group consisting of a non-steroidal anti-inflammatory drug(NSAID), an antibiotic, an antifungal a steroid, an anti-cancer agent,and osteogenic factor and a bone resorption inhibitor; (ii) aphosphatidylethanolamine; and (iii) a phosphatidylcholine; and (c)mixing and homogenizing the products resulting from steps (a) and (b).In another embodiment, the phosphatidylethanolamine is included in thenon-polar, volatile organic solvent instead of the water-miscible,volatile organic solvent. In another embodiment, the biodegradablepolyester is selected from the group consisting of PLA, PGA and PLGA. Inanother embodiment, the biodegradable polymer is any other suitablebiodegradable polyester known in the art. In another embodiment, themixture containing the non-polar, organic solvent is homogenized priorto mixing it with the mixture organic solvent. In another embodiment,the mixture containing the water-miscible, organic solvent ishomogenized prior to mixing it with the mixture containing thenon-polar, organic solvent. In another embodiment, the polymer in themixture of step (a) is lipid saturated. In another embodiment, thematrix composition is lipid saturated. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the matrix composition of the present inventioncan be used for coating fully or partially the surface of differentsubstrates. In another embodiment substrates to be coated include atleast one material selected from the group consisting of carbon fibers,stainless steel, cobalt-chromium, titanium alloy, tantalum, ceramic andcollagen or gelatin. In another embodiment substrates may include anymedical devices such as orthopedic nails, orthopedic screws, orthopedicstaples, orthopedic wires and orthopedic pins used in orthopedicsurgery, metal or polymeric implants used in both orthopedic andperiodontal surgery, bone filler particles and absorbable gelatinsponge. Bone filler particles can be any one of allogeneic (i.e., fromhuman sources), xenogeneic (i.e., from animal sources) and artificialbone particles. In another embodiment a treatment using the coatedsubstrates and administration of the coated substrates will followprocedures known in the art for treatment and administration of similaruncoated substrates. In another embodiment bone filler particles coatedwith the biodegradable matrix of the present invention are administeredsubstantially as a single ingredient (not administered as part of amixture with other ingredients). Alternatively, the coated bone fillerparticles are mixed with any other commercially available bone fillerparticles or autologous bone before administration. In anotherembodiment, the mixture of bone filler particles comprises at least oneof: non-coated particles, particles coated with matrix compositionsincorporating a pharmaceutically active agent, particles coated withmatrix compositions incorporating a plurality of pharmaceutically activeagents or a combination thereof. In another embodiment the amounts,ratios and types of ingredients forming the matrix composition of thepresent invention are varied so to adjust the polymer-lipid basis to thebiophysical/biochemical properties of the pharmaceutically active agent,the therapeutically effective dose of the pharmaceutically active agentand to the desired sustained release time period (typically in the rangeof days to months).

It is to be emphasized that the sustained release period using thecompositions of the present invention can be programmed taking intoaccount two major factors: (i) the weight ratio between the polymer andthe lipid content, specifically the phospholipid having fatty acidmoieties of at least 14 carbons, and (ii) the biochemical and/orbiophysical properties of the biopolymer and the lipid. Specifically,the degradation rate of the polymer and the fluidity of the lipid shouldbe considered. For example, a PLGA (85:15) polymer will degrade slowerthan a PLGA (50:50) polymer. A phosphatidylcholine (14:0) is more fluid(less rigid and less ordered) at body temperature than aphosphatidylcholine (18:0). Thus, for example, the release rate of adrug incorporated in a matrix composition comprising PLGA (85:15) andphosphatidylcholine (18:0) will be slower than that of a drugincorporated in a matrix composed of PLGA (50:50) andphosphatidylcholine (14:0). Another aspect that will determine therelease rate is the physical characteristics of the entrapped orimpregnated drug. In addition, the release rate of drugs can further becontrolled by the addition of other lipids into the formulation of thesecond solution. This can includes fatty acids of different length suchas lauric acid (C12:0), membrane active sterols (such as cholesterol) orother phospholipids such as phosphatidylethanolamine. According tovarious embodiments the active agent is released from the compositionover a desired period ranging between several days to several months.

These and other features and advantages of the present invention willbecome more readily understood and appreciated from the detaileddescription of the invention that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 (A and B): Doxycycline hyclate (DOX) encapsulated in theformulation of the present invention is being released constantly for atleast 3 weeks. A) DOX released from a matrix comprising PLGA 85:15,cholesterol, alpha-tocopherol, and DSPC (18:0); B) DOX released from amatrix comprising PLGA 85:15, cholesterol, and DSPC (18:0).

FIG. 2: The majority (−70%) of DOX encapsulated in the formulation ofthe present invention is being released following zero order kinetics. Yaxis represents the velocity of the release of DOX in μg/ml/hour.

FIG. 3: The macro-structure of the bone particles is not affected bycoating with the formulation of the present invention. (A) the originalstructure of bone particles; (B) bone particles coated with theformulation of the present invention encapsulating DOX; (C) the boneparticles of (B) after 60 days of incubation in serum.

FIG. 4: The matrix formulation coating the surface of bone particlesundergoes gradual surface degradation. (A) untreated surface; (B)surface of a coated bone particle; (C) the surface of a coated boneparticle after 1 day in 10% FBS at 37° C.; (D) the surface of coatedbone particle after 30 days in 10% FBS at 37° C.; (E) surface of coatedbone particle after 60 days in 10% FBS at 37° C.

FIG. 5: The ordered structured of the matrix formulation (PLGA (85:15),DPPC (16:0), Cholesterol 10%) is demonstrated by electron microscopy (X18,000). The bright colored lines represent the polymer, where as thelipid is represented by the dark filling in between the polymericmaterial.

FIG. 6: The specific polymer/lipid compositions of the formulations ofthe present invention determine the release rate of a given drug. Theinfluence of various concentrations of lauric acid (LA) andphosphatidylethanolamine (PE) given in w/w % of the formulation on therelease period of 90% of the entrapped drug.

FIG. 7: The use of dimethyl phosphatidylethanolamine (DMPE) vs.cholesterol in formulations of the present invention. Comparison of therelease profile of DOX from bone particles coated with a formulationthat comprises dimethyl dimyristoyl phosphatidylethanolamine (DMPE)(diamonds) in the first organic solution during preparation versus aformulation that comprises cholesterol (squares) at the same preparationstage following hydration of the bone particles (5% serum at 37° C.).

FIG. 8: The nature of the polymer and phospholipid determines therelease rate of the entrapped pharmaceutical active agent. Flurbiprofenis released from a matrix comprising PLGA (50:50) and DMPC (14:0),whereas doxycycline hyclate is released from a matrix compositioncomprising PLGA (85:15) and DSPC (18:0).

FIG. 9: The release profile of DOX from bone particles coated with aformulation that contains DOX and mixed with similar not-coated boneparticles in a ratio of 1:4 (diamonds) versus the release profile offree DOX mixed with the same amount of not-coated bone particlesfollowing bone particles hydration (5% serum at 37° C.).

FIG. 10: The duration of drug release from bone particles coated withthe formulations of the invention is linearly depended on theformulation mass. Bone particles (12 mg/sample) were coated withdifferent mass of formulation containing DOX (X-axis reflecting theformulation mass in mg). Following bone particles hydration, the releaseof DOX from the formulation was monitored. Y-axis is reflecting the dayin which the accumulated release of the entrapped DOX exceeded 90% ofthe overall entrapped dose.

FIG. 11: The release profile of the antifungal drug, Thiobendazole(TBZ), from bone particles coated with a formulation (PLGA 50:50,cholesterol and DMPC (14:0) that contains TBZ (10% of the total mass ofthe formulation).

FIG. 12: Antibiotic release from an absorbable gelatin sponge (Gelatamp.ROEKO) coated with the matrix formulation of the invention (PLGA 75:25,PC 16:0, cholesterol 10% and DOX 10%). The release of DOX from anabsorbable gelatin sponge pre-wetted with DOX solution having a similardrug dose served as control.

FIG. 13: The degradation of the bone particle coating formulation asreflected by surface element analysis. Following hydration of the coatedbone particles, the percentages of carbon, calcium and phosphate atomson the surface of the coated bone particles were monitored by SEM. TheX-axis is presenting the time post hydration of the coated bone samples.

FIG. 14: Turbidity analysis of the supernatant solution (5% serum) ofhydrated bone particles: 4 different types of bone particles wereanalyzed: (i) plain, non-coated bone particles (ii) bone particlescoated with the matrix composition of the invention having DOX as thepharmaceutically active agent (iii) bone particles coated with DPPC andDOX and (iv) bone particles coated with PLGA. The turbidities of thesupernatants into which the bone particles were immersed were measured 1hour after hydration and at 37° C. (A). After an hour of incubation thehydration medium was replaced by a fresh medium and turbidity wasmeasured after 23 hours of incubation at 37° C. (B). An electronmicroscopy image of the hydration solution into which bone particlescoated with the matrix formulation of the invention were immersed, taken24 hours after hydration at 37° C. (C). Size distribution (D) and zetapotential (E) analysis of the material released from hydrated boneparticles.

FIG. 15: Encapsulated DOX and fluorescently labeled phosphatidylcholine(NBD-PC) are co-released from the surface of coated bone particles intothe surrounding medium (5% FBS at 37° C.) following zero order kinetics.

FIG. 16: Small Angle X-ray Scattering (SAXS) analysis of bone particlescoated with the matrix formulation of the invention (PLGA 85:15, DPPC16:0, DOX) reveals the matrix has an ordered structure. As controls, thescattering profile of dried DOPS (18:1) powder and plain, non-coatedbone particles were recorded.

FIG. 17: A. Differential scanning calorimetric analysis (DSC) suggestscholesterol decreases the heat intake by PLGA upon heating. B. PLGA heatuptake decrease was evident in the presence of other lipids such as theantioxidant α-tocopherol, but not with lipids such as mineral oil(containing alkanes with carbon chain of C12-C18).

FIG. 18: A dental metal implant made of titanium coated with a matrixformulation comprising PLGA (18:15), DSPC (18:0) cholesterol 10% and 10%DOX. A. The uncoated dental implant. B. the coated implant. The brightcolor of the coated implant under UV light is due to the fluorescenceemission of DOX.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions for extended release of anactive ingredient, comprising a lipid-based matrix comprising abiodegradable polymer. The present invention also provides methods ofproducing the matrix compositions and methods for using the matrixcompositions to provide controlled release of an active ingredient inthe body of a subject in need thereof.

The term “controlled release” refers to control of the rate and/orquantity of pharmaceutically active agent(s) delivered by the matrixcompositions of the invention. The controlled release can be continuousor discontinuous, and/or linear or non-linear.

The term “sustained release” means that pharmaceutical active agent isreleased over an extended period of time.

In certain embodiments, the present invention provides a matrixcomposition comprising: (a) biodegradable polyester; (b) aphosphoglyceride having hydrocarbon moieties of at least 14 carbons; and(c) a pharmaceutical active agent. According to some embodiments thepharmaceutical agent is selected from the group consisting of anantibiotic, an antifungal, an NSAID, a steroid, an anticancer agent, anosteogenic factor and a bone resorption inhibitor.

In certain embodiments the phosphoglyceride is a phospholipid. In someembodiments, the phospholipid is a phosphatidylcholine having fatty acidmoieties of at least 14 carbons. In another embodiment, the compositionfurther comprises a phosphatidylethanolamine having a fatty acidmoieties of at least 14 carbons. In another embodiment, the compositionfurther comprises a sterol. In some embodiments the sterol ischolesterol.

In another embodiment, the matrix composition is lipid saturated. “Lipidsaturated,” as used herein, refers to saturation of the polymer of thematrix composition with lipids including phospholipids, in combinationwith any hydrophobic drug and targeting moiety present in the matrix,and any other lipids that may be present. The matrix composition issaturated by whatever lipids are present. Lipid-saturated matrices ofthe present invention exhibit the additional advantage of not requiringa synthetic emulsifier or surfactant such as polyvinyl alcohol; thus,compositions of the present invention are typically substantially freeof polyvinyl alcohol. Methods for determining the polymer:lipid ratio toattain lipid saturation and methods of determining the degree of lipidsaturation of a matrix are described hereinbelow.

In another embodiment, the matrix composition is homogeneous. In anotherembodiment, the matrix composition is in the form of a lipid-saturatedmatrix whose shape and boundaries are determined by the biodegradablepolymer. In another embodiment, the matrix composition is in the form ofan implant. Preferably, the polyester, the phosphatidylethanolamine, andthe sterol are incorporated into the matrix composition. In anotherembodiment, the phosphatidylcholine is also incorporated into the matrixcomposition. In another embodiment, the antibiotic is also incorporatedinto the matrix composition. In another embodiment, the antibiotic haslow water solubility. In another embodiment, the antibiotic is ahydrophobic antibiotic. In another embodiment, the antibiotic is anamphipathic antibiotic. In another embodiment, the composition furthercomprises a non-steroidal anti-inflammatory drug (NSAID). In anotherembodiment, the NSAID as well is incorporated into the matrixcomposition. In another embodiment, the NSAID has low water solubility.Each possibility represents a separate embodiment of the presentinvention.

In one embodiment, the present invention provides a matrix compositioncomprising: (a) biodegradable polyester; (b) a sterol; (c) aphosphatidylethanolamine having a fatty acid moieties of at least 14carbons; (d) a phosphatidylcholine having a fatty acid moieties of atleast 14 carbons; and (e) an antibiotic or an antifungal. In anotherembodiment, the matrix composition is lipid saturated. Preferably, thepolyester, the phosphatidylethanolamine, and the sterol are incorporatedinto the matrix composition. In another embodiment, thephosphatidylcholine is also incorporated into the matrix composition. Inanother embodiment, the antibiotic is also incorporated into the matrixcomposition. In another embodiment, the antibiotic has low watersolubility. In another embodiment, the antibiotic is a hydrophobicantibiotic. In another embodiment, the antibiotic is an amphipathicantibiotic. In another embodiment, the composition further comprises anon-steroidal anti-inflammatory drug (NSAID). In another embodiment, theNSAID as well is incorporated into the matrix composition. In anotherembodiment, the NSAID has low water solubility. In another embodiment,the matrix composition is in the form of a lipid-saturated matrix whoseshape and boundaries are influenced by the nature of the biodegradablepolymer. In another embodiment, the matrix composition is in the form ofan implant. In another embodiment, the matrix composition ishomogeneous. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a matrixcomposition comprising: (a) biodegradable polyester; (b) a sterol; (c) aphosphatidylethanolamine having fatty acid moieties of at least 14carbons; (d) a phosphatidylcholine having fatty acid moieties of atleast 14 carbons; (e) a non-steroidal anti-inflammatory drug (NSAID). Inanother embodiment, the matrix composition is lipid saturated.Preferably, the polyester, the phosphatidylethanolamine, and the sterolare incorporated into the matrix composition. In another embodiment, thephosphatidylcholine is also incorporated into the matrix composition. Inanother embodiment, the NSAID is also incorporated into the matrixcomposition. In another embodiment, the NSAID has low water solubility.In another embodiment, the NSAID is a hydrophobic NSAID. In anotherembodiment, the NSAID is an amphipathic NSAID. In another embodiment,the matrix composition is in the form of a lipid-saturated matrix whoseshape and boundaries are determined by the biodegradable polymer. Inanother embodiment, the matrix composition is in the form of an implant.In another embodiment, the matrix composition is homogeneous. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a matrixcomposition comprising: (a) biodegradable polyester; (b) a sterol; (c) aphosphatidylethanolamine having fatty acid moieties of at least 14carbons; (d) a phosphatidylcholine having fatty acid moieties of atleast 14 carbons; and (e) an osteogenic factor or a bone resorptioninhibitor. In another embodiment, the matrix composition is lipidsaturated. Preferably, the polyester, the phosphatidylethanolamine, andthe sterol are incorporated into the matrix composition. In anotherembodiment, the phosphatidylcholine is also incorporated into the matrixcomposition. In another embodiment, the bone resorption inhibitor isalso incorporated into the matrix composition. In another embodiment,the bone resorption inhibitor has low water solubility. In anotherembodiment, the bone resorption inhibitor is a hydrophobic boneresorption inhibitor. In another embodiment, the bone resorptioninhibitor is an amphipathic bone resorption inhibitor. In anotherembodiment, the composition further comprises an NSAID. In anotherembodiment, the NSAID as well is incorporated into the matrixcomposition. In another embodiment, the matrix composition is in theform of a lipid-saturated matrix whose shape and boundaries aredetermined by the biodegradable polymer. In another embodiment, thematrix composition is in the form of an implant. In another embodiment,the matrix composition is homogeneous. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides a matrixcomposition comprising: (a) biodegradable polyester; (b) a sterol; (c) aphosphatidylethanolamine having fatty acid moieties of at least 14carbons; (d) a phosphatidylcholine having fatty acid moieties of atleast 14 carbons; (e) an active agent; and (f) a targeting moietycapable of interacting with a surface molecule of a target cell, atarget molecule or a target surface. In another embodiment, the matrixcomposition is lipid saturated. In another embodiment, the active agentis selected from the group consisting of an NSAID, an antibiotic, and abone resorption inhibitor. In another embodiment, the polymer and thephospholipid form the matrix composition that is substantially free ofwater. In another embodiment, the active agent and the targeting moietyare integrated into the lipid vesicle. In another embodiment, the matrixcomposition is in the form of a lipid-saturated matrix whose shape andboundaries are determined by the biodegradable polymer. In anotherembodiment, the matrix composition is in the form of an implant. Inanother embodiment, the matrix composition is homogeneous. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the biodegradable polyester of methods andcompositions of the present invention is associated with the sterol viahydrogen bonds.

As provided herein, the matrix composition of methods and compositionsof the present invention is capable of being molded intothree-dimensional configurations of varying thickness and shape.Accordingly, the matrix formed can be produced to assume a specificshape including a sphere, cube, rod, tube, sheet, or into strings. Inthe case of freeze-drying, the shape is determined by the shape of amold or support which may be made of any inert material and may be incontact with the matrix on all sides, as for a sphere or cube, or on alimited number of sides as for a sheet. The matrix may be shaped in theform of body cavities as required for implant design. Removing portionsof the matrix with scissors, a scalpel, a laser beam or any othercutting instrument can create any refinements required in thethree-dimensional structure. Each possibility represents a separateembodiment of the present invention.

Advantageously, the matrix compositions of the present invention areprepared by methods which do not involve the formation of emulsions, andmay avoid the use of aqueous media altogether. The generation ofemulsions that are subsequently dried necessarily results in vesicles ormicrospheres. In contrast, the matrices produced without aqueous mediaform homogeneous liquid mixtures that can be molded or formed into threedimensional articles of any shape or can coat the surface of differentsubstrates. In order to produce molded or coated articles the mixture ofpolymer and lipids and active ingredients within the appropriateselected volatile organic solvents will be used to coat the desiredsurface or to fit the desired shape.

The matrix composition of methods and compositions of the presentinvention is capable of coating the surface of different substrates.Substrates to be coated include materials selected from the groupconsisting of carbon fibers, stainless steel, cobalt-chromium, titaniumalloy, tantalum, ceramic and collagen or gelatin. Specifically,substrates may include any medical devices such as orthopedic nails,orthopedic screws, orthopedic staples, orthopedic wires and orthopedicpins used in orthopedic surgery, metal or polymeric implants used inboth orthopedic and periodontal surgery, bone filler particles andabsorbable gelatin sponge. Bone filler particles can be selected fromany one of allogeneic (i.e., from human sources), xenogeneic (i.e., fromanimal sources) and artificial bone particles.

According to some embodiments, the matrix composition of the presentinvention is useful as a bone graft material. This term refers to anatural or synthetic material that supports attachment of newosteoblasts and osteoprogenitor cells or can induce non-differentiatedstem cells or osteoprogenitor cells to differentiate into osteoblasts.In another embodiment, the bone graft material is selected from thegroup consisting of an allograft, an alloplast, a xenograft, and anautologous bone graft. In other example the lipid matrix of the presentinvention can also be used in conjunction with a collagen membrane orcollagen sponge or gelatin sponge or the like.

Lipids

“Phospholipids” are phosphoglycerides having a single phosphatidyllinkage on a glycerol backbone and fatty acids at the remaining twopositions. However, it is to be understood explicitly thatphosphoglycerides having hydrocarbon chains other than fatty acidresidues including alkyl chains, alkenyl chains or any other hydrocarbonchain of at least 14 carbons are included within the scope of thepresent invention. The linkage may be an ether linkage instead of anacyl linkage found in phospholipids.

“Phosphatidylcholine” refers to a phosphoglyceride having aphosphorylcholine head group. Phosphatidylcholine compounds, in anotherembodiment, have the following structure:

The R and R′ moieties are fatty acids, typically naturally occurringfatty acids or derivatives of naturally occurring fatty acids. In someembodiments, the fatty acid moieties are saturated fatty acid moieties.In some embodiments, the fatty acid moieties are unsaturated fatty acidmoieties. “Saturated”, refers to the absence of a double bond in thehydrocarbon chain. In another embodiment, the fatty acid moieties haveat least 14 carbon atoms. In another embodiment, the fatty acid moietieshave 16 carbon atoms. In another embodiment, the fatty acid moietieshave 18 carbon atoms. In another embodiment, the fatty acid moietieshave 16-18 carbon atoms. In another embodiment, the fatty acid moietiesare chosen such that the gel-to-liquid-crystal transition temperature ofthe resulting matrix is at least 40° C. In another embodiment, the fattyacid moieties are both palmitoyl. In another embodiment, the fatty acidmoieties are both stearoyl. In another embodiment, the fatty acidmoieties are both arachidoyl. In another embodiment, the fatty acidmoieties are palmitoyl and stearoyl. In another embodiment, the fattyacid moieties are palmitoyl and arachidoyl. In another embodiment, thefatty acid moieties are arachidoyl and stearoyl. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the phosphatidylcholine is a naturally-occurringphosphatidylcholine. In another embodiment, the phosphatidylcholine is asynthetic phosphatidylcholine. In another embodiment, thephosphatidylcholine contains a naturally-occurring distribution ofisotopes. In another embodiment, the phosphatidylcholine is a deuteratedphosphatidylcholine. In another embodiment, the phosphatidylcholine islabeled with any other isotope or label. Preferably, thephosphatidylcholine is a symmetric phosphatidylcholine (i.e. aphosphatidylcholine wherein the two fatty acid moieties are identical).In another embodiment, the phosphatidylcholine is an asymmetricphosphatidylcholine.

Non-limiting examples of phosphatidylcholines are1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),dioleoyl-phosphatidylcholine (DOPC),1-palmitoyl-2-oleoyl-phosphatidylcholine, and phosphatidylcholinesmodified with any of the fatty acid moieties enumerated hereinabove. Inanother embodiment, the phosphatidylcholine is selected from the groupconsisting of DSPC and DOPC, and1-palmitoyl-2-oleoyl-phosphatidylcholine.

In another embodiment, the phosphatidylcholine is any otherphosphatidylcholine known in the art. Each phosphatidylcholinerepresents a separate embodiment of the present invention.

“Phosphatidylethanolamine” refers to a phosphoglyceride having aphosphoryl ethanolamine head group. Phosphatidylethanolamine compounds,in another embodiment, have the following structure:

The R₁ and R₂ moieties are fatty acids, typically naturally occurringfatty acids or derivatives of naturally occurring fatty acids. Inanother embodiment, the fatty acid moieties are saturated fatty acidmoieties. “Saturated” in another embodiment, refers to the absence of adouble bond in the hydrocarbon chain. In another embodiment, the fattyacid moieties have at least 14 carbon atoms. In another embodiment, thefatty acid moieties have at least 16 carbon atoms. In anotherembodiment, the fatty acid moieties have 14 carbon atoms. In anotherembodiment, the fatty acid moieties have 16 carbon atoms. In anotherembodiment, the fatty acid moieties have 18 carbon atoms. In anotherembodiment, the fatty acid moieties have 14-18 carbon atoms. In anotherembodiment, the fatty acid moieties have 14-16 carbon atoms. In anotherembodiment, the fatty acid moieties have 16-18 carbon atoms. In anotherembodiment, the fatty acid moieties are chosen such that thegel-to-liquid-crystal transition temperature of the resulting matrix isat least 40° C. In another embodiment, the fatty acid moieties are bothmyristoyl. In another embodiment, the fatty acid moieties are bothpalmitoyl. In another embodiment, the fatty acid moieties are bothstearoyl. In another embodiment, the fatty acid moieties are botharachidoyl. In another embodiment, the fatty acid moieties are myristoyland stearoyl. In another embodiment, the fatty acid moieties aremyristoyl and arachidoyl. In another embodiment, the fatty acid moietiesare myristoyl and palmitoyl. In another embodiment, the fatty acidmoieties are palmitoyl and stearoyl. In another embodiment, the fattyacid moieties are palmitoyl and arachidoyl. In another embodiment, thefatty acid moieties are arachidoyl and stearoyl. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the phosphatidylethanolamine is anaturally-occurring phosphatidylethanolamine. In another embodiment, thephosphatidylethanolamine is a synthetic phosphatidylethanolamine. Inanother embodiment, the phosphatidylethanolamine is a deuteratedphosphatidylethanolamine. In another embodiment, thephosphatidylethanolamine is labeled with any other isotope or label. Inanother embodiment, the phosphatidylethanolamine contains anaturally-occurring distribution of isotopes. Preferably, thephosphatidylethanolamine is a symmetric phosphatidylethanolamine. Inanother embodiment, the phosphatidylethanolamine is an asymmetricphosphatidylethanolamine.

Non-limiting examples of phosphatidylethanolamines are dimethyldimyristoyl phosphatidylethanolamine (DMPE) anddipalmitoyl-phosphatidylethanolamine (DPPE), andphosphatidylethanolamines modified with any of the fatty acid moietiesenumerated hereinabove. In another embodiment, thephosphatidylethanolamine is selected from the group consisting of DMPEand DPPE.

In another embodiment, the phosphatidylethanolamine is any otherphosphatidylethanolamine known in the art. Each phosphatidylethanolaminerepresents a separate embodiment of the present invention.

“Sterol” in one embodiment refers to a steroid with a hydroxyl group atthe 3-position of the A-ring. In another embodiment, the term refers toa steroid having the following structure:

In another embodiment, the sterol of methods and compositions of thepresent invention is a zoosterol. In another embodiment, the sterol ischolesterol:

In another embodiment, the sterol is any other zoosterol known in theart. In another embodiment, the moles of sterol are up to 40% of themoles of total lipids present. In another embodiment, the sterol isincorporated into the matrix composition. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the cholesterol is present in an amount of 10-50percentage of the total weight of lipid content of the matrixcomposition. In another embodiment, the weight percentage is 20-50%. Inanother embodiment, the weight percentage is 10-40%. In anotherembodiment, the weight percentage is 30-50%. In another embodiment, theweight percentage is 20-60%. In another embodiment, the weightpercentage is 25-55%. In another embodiment, the weight percentage is35-55%. In another embodiment, the weight percentage is 30-60%. Inanother embodiment, the weight percentage is 30-55%. In anotherembodiment, the weight percentage is 20-50%. In another embodiment, theweight percentage is 25-55%. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a composition of the present invention furthercomprises a lipid other than phosphatidylcholine,phosphatidylethanolamine, or a sterol. In another embodiment, theadditional lipid is a phosphoglyceride. In another embodiment, theadditional lipid is selected from the group consisting of aphosphatidylserine, a phosphatidylglycerol, and a phosphatidylinositol.In another embodiment, the additional lipid is selected from the groupconsisting of a phosphatidylserine, a phosphatidylglycerol, aphosphatidylinositol, and a sphingomyelin. In another embodiment, acombination of any 2 or more of the above additional lipids is present.In another embodiment, the polymer, phosphatidylcholine,phosphatidylethanolamine, sterol, and additional lipid(s) are allincorporated into the matrix composition. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, phosphatidylcholine(s) (PC) compose at least 30%of the total lipid content of the matrix composition. In anotherembodiment, PC(s) compose at least 35% of the total lipid content. Inanother embodiment, PC(s) compose at least 40% of the total lipidcontent. In another embodiment, PC(s) compose at least 45% of the totallipid content. In another embodiment, PC(s) compose at least 50% of thetotal lipid content. In another embodiment, PC(s) compose at least 55%of the total lipid content. In another embodiment, PC(s) compose atleast 60% of the total lipid content. In another embodiment, PC(s)compose at least 65% of the total lipid content. In another embodiment,PC(s) compose at least 70% of the total lipid content. In anotherembodiment, PC(s) compose at least 75% of the total lipid content. Inanother embodiment, PC(s) compose at least 80% of the total lipidcontent. In another embodiment, PC(s) compose at least 85% of the totallipid content. In another embodiment, PC(s) compose at least 90% of thetotal lipid content. In another embodiment, PC(s) compose at least 95%of the total lipid content. In another embodiment, PC(s) compose over95% of the total lipid content. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a composition of the present invention furthercomprises a phosphatidylserine. “Phosphatidylserine” refers to aphosphoglyceride having a phosphorylserine head group.Phosphatidylserine compounds, in another embodiment, have the followingstructure:

The R₁ and R₂ moieties are fatty acids, typically naturally occurringfatty acids or derivatives of naturally occurring fatty acids. Inanother embodiment, the fatty acid moieties are saturated fatty acidmoieties. In another embodiment, the fatty acid moieties have at least14 carbon atoms. In another embodiment, the fatty acid moieties have atleast 16 carbon atoms. In another embodiment, the fatty acid moietiesare chosen such that the gel-to-liquid-crystal transition temperature ofthe resulting matrix is at least 40° C. In another embodiment, the fattyacid moieties are both myristoyl. In another embodiment, the fatty acidmoieties are both palmitoyl. In another embodiment, the fatty acidmoieties are both stearoyl. In another embodiment, the fatty acidmoieties are both arachidoyl. In another embodiment, the fatty acidmoieties are myristoyl and stearoyl. In another embodiment, the fattyacid moieties are a combination of two of the above fatty acid moieties.

In another embodiment, the phosphatidylserine is a naturally-occurringphosphatidyl serine. In another embodiment, the phosphatidylserine is asynthetic phosphatidyl serine. In another embodiment, thephosphatidylserine is a deuterated phosphatidyl serine. In anotherembodiment, the phosphatidylserine is labeled with any other isotope orlabel. In another embodiment, the phosphatidylserine contains anaturally-occurring distribution of isotopes. In another embodiment, thephosphatidylserine is a symmetric phosphatidylserine. In anotherembodiment, the phosphatidylserine is an asymmetric phosphatidylserine.

Non-limiting examples of phosphatidylserines are phosphatidylserinesmodified with any of the fatty acid moieties enumerated hereinabove. Inanother embodiment, the phosphatidylserine is any otherphosphatidylserine known in the art. Each phosphatidylserine representsa separate embodiment of the present invention.

In another embodiment, a composition of the present invention furthercomprises a phosphatidylglycerol. “Phosphatidylglycerol” refers to aphosphoglyceride having a phosphoryl glycerol head group.Phosphatidylglycerol compounds, in another embodiment, have thefollowing structure:

The 2 bonds to the left are connected to fatty acids, typicallynaturally occurring fatty acids or derivatives of naturally occurringfatty acids. In another embodiment, the phosphatidylglycerol is anaturally-occurring phosphatidylglycerol. In another embodiment, thephosphatidylglycerol is a synthetic phosphatidyl glycerol. In anotherembodiment, the phosphatidylglycerol is a deuteratedphosphatidylglycerol. In another embodiment, the phosphatidylglycerol islabeled with any other isotope or label. In another embodiment, thephosphatidylglycerol contains a naturally-occurring distribution ofisotopes. In another embodiment, the phosphatidylglycerol is a symmetricphosphatidylglycerol. In another embodiment, the phosphatidylglycerol isan asymmetric phosphatidylglycerol. In another embodiment, the termincludes diphosphatidylglycerol compounds having the followingstructure:

The R and R′ moieties are fatty acids, typically naturally occurringfatty acids or derivatives of naturally occurring fatty acids. Inanother embodiment, the fatty acid moieties are saturated fatty acidmoieties. In another embodiment, the fatty acid moieties have at least14 carbon atoms. In another embodiment, the fatty acid moieties have atleast 16 carbon atoms. In another embodiment, the fatty acid moietiesare chosen such that the gel-to-liquid-crystal transition temperature ofthe resulting matrix is at least 40° C. In another embodiment, the fattyacid moieties are both myristoyl. In another embodiment, the fatty acidmoieties are both palmitoyl. In another embodiment, the fatty acidmoieties are both stearoyl. In another embodiment, the fatty acidmoieties are both arachidoyl. In another embodiment, the fatty acidmoieties are myristoyl and stearoyl. In another embodiment, the fattyacid moieties are a combination of two of the above fatty acid moieties.

Non-limiting examples of phosphatidylglycerols are phosphatidylglycerolsmodified with any of the fatty acid moieties enumerated hereinabove. Inanother embodiment, the phosphatidylglycerol is any otherphosphatidylglycerol known in the art. Each phosphatidylglycerolrepresents a separate embodiment of the present invention.

In another embodiment, a composition of the present invention furthercomprises a phosphatidylinositol. “Phosphatidyl inositol” refers to aphosphoglyceride having a phosphorylinositol head group.Phosphatidylinositol compounds, in another embodiment, have thefollowing structure:

The R₁ and R₂ moieties are fatty acids, typically naturally occurringfatty acids or derivatives of naturally occurring fatty acids. Inanother embodiment, the fatty acid moieties are saturated fatty acidmoieties. In another embodiment, the fatty acid moieties have at least14 carbon atoms. In another embodiment, the fatty acid moieties have atleast 16 carbon atoms. In another embodiment, the fatty acid moietiesare chosen such that the gel-to-liquid-crystal transition temperature ofthe resulting matrix is at least 40° C. In another embodiment, the fattyacid moieties are both myristoyl. In another embodiment, the fatty acidmoieties are both palmitoyl. In another embodiment, the fatty acidmoieties are both stearoyl. In another embodiment, the fatty acidmoieties are both arachidoyl. In another embodiment, the fatty acidmoieties are myristoyl and stearoyl. In another embodiment, the fattyacid moieties are a combination of two of the above fatty acid moieties.

In another embodiment, the phosphatidyl inositol is anaturally-occurring phosphatidylinositol. In another embodiment, thephosphatidylinositol is a synthetic phosphatidylinositol. In anotherembodiment, the phosphatidylinositol is a deuteratedphosphatidylinositol. In another embodiment, the phosphatidylinositol islabeled with any other isotope or label. In another embodiment, thephosphatidylinositol contains a naturally-occurring distribution ofisotopes. In another embodiment, the phosphatidylinositol is a symmetricphosphatidylinositol. In another embodiment, the phosphatidylinositol isan asymmetric phosphatidylinositol.

Non-limiting examples of phosphatidylinositols are phosphatidylinositolsmodified with any of the fatty acid moieties enumerated hereinabove. Inanother embodiment, the phosphatidylinositol is any otherphosphatidylinositol known in the art. Each phosphatidylinositolrepresents a separate embodiment of the present invention.

In another embodiment, a composition of the present invention furthercomprises a sphingolipid. In another embodiment, the sphingolipid isceramide. In another embodiment, the sphingolipid is a sphingomyelin.“Sphingomyelin” refers to a sphingosine-derived phospholipid.Sphingomyelin compounds, in another embodiment, have the followingstructure:

The R moiety is a fatty acid, typically a naturally occurring fatty acidor a derivative of a naturally occurring fatty acid. In anotherembodiment, the sphingomyelin is a naturally-occurring sphingomyelin. Inanother embodiment, the sphingomyelin is a synthetic sphingomyelin. Inanother embodiment, the sphingomyelin is a deuterated sphingomyelin. Inanother embodiment, the sphingomyelin is labeled with any other isotopeor label. In another embodiment, the sphingomyelin contains anaturally-occurring distribution of isotopes.

In another embodiment, the fatty acid moiety of a sphingomyelin ofmethods and compositions of the present invention has at least 14 carbonatoms. In another embodiment, the fatty acid moiety has at least 16carbon atoms. In another embodiment, the fatty acid moiety is chosensuch that the gel-to-liquid-crystal transition temperature of theresulting matrix is at least 40° C.

Non-limiting examples of sphingomyelins are sphingomyelins modified withany of the fatty acid moieties enumerated hereinabove. In anotherembodiment, the sphingomyelin is any other sphingomyelin known in theart. Each sphingomyelin represents a separate embodiment of the presentinvention.

“Ceramide” refers to a compound having the structure:

The R moiety is a fatty acid typically naturally occurring fatty acid orderivatives of naturally occurring fatty acids. In another embodiment,the fatty acid is a longer-chain (to C₂₄ or greater). In anotherembodiment, the fatty acids are saturated fatty acids. In anotherembodiment, the fatty acids are monoenoic fatty acids. In anotherembodiment, the fatty acids are n-9 monoenoic fatty acids. In anotherembodiment, the fatty acids contain a hydroxyl group in position 2. Inanother embodiment, the fatty acids are other suitable fatty acids knownin the art. In another embodiment, the ceramide is a naturally-occurringceramide. In another embodiment, the ceramide is a synthetic ceramide.In another embodiment, the ceramide is incorporated into the matrixcomposition. Each possibility represents a separate embodiment of thepresent invention.

Each sphingolipid represents a separate embodiment of the presentinvention.

In another embodiment, a composition of the present invention furthercomprises a pegylated lipid. In another embodiment, the PEG moiety has aMW of 500-5000 daltons. In another embodiment, the PEG moiety has anyother suitable MW. Non-limiting examples of suitable PEG-modified lipidsinclude PEG moieties with a methoxy end group, e.g. PEG-modifiedphosphatidylethanolamine and phosphatidic acid (structures A and B),PEG-modified diacylglycerols and dialkylglycerols (structures C and D),PEG-modified dialkylamines (structure E) and PEG-modified1,2-diacyloxypropan-3-amines (structure F) as depicted below. In anotherembodiment, the PEG moiety has any other end group used in the art. Inanother embodiment, the pegylated lipid is selected from the groupconsisting of a PEG-modified phosphatidylethanolamine, a PEG-modifiedphosphatidic acid, a PEG-modified diacylglycerol, a PEG-modifieddialkylglycerol, a PEG-modified dialkylamine, and a PEG-modified1,2-diacyloxypropan-3-amine. In another embodiment, the pegylated lipidis any other pegylated phospholipid known in the art. Each possibilityrepresents a separate embodiment of the present invention.

Preferably, the pegylated lipid is present in an amount of less than 10mole percent of total lipids in the matrix composition. In anotherembodiment, the percentage is less than 9 mole % of the total lipids. Inanother embodiment, the percentage is less than 8 mole %. In anotherembodiment, the percentage is less than 7 mole %. In another embodiment,the percentage is less than 6 mole %. In another embodiment, thepercentage is less than 5 mole %. In another embodiment, the percentageis less than 4 mole %. In another embodiment, the percentage is lessthan 3 mole %. In another embodiment, the percentage is less than 2 mole%. In another embodiment, the percentage is less than 1 mole %. Eachpossibility represents a separate embodiment of the present invention.

Polymers

The biodegradable polyester of methods and compositions of the presentinvention is, in another embodiment, PLA (polylactic acid). “PLA” refersto poly(L-lactide), poly(D-lactide), and poly(DL-lactide). Arepresentative structure of poly(DL-lactide) is depicted below:

In another embodiment, the polymer is PGA (polyglycolic acid). Inanother embodiment, the polymer is PLGA (poly(lactic-co-glycolic acid).The PLA contained in the PLGA may be any PLA known in the art, e.g.either enantiomer or a racemic mixture. A representative structure ofPLGA is depicted below:

The PLGA of methods and compositions of the present invention has, inanother embodiment, a 1:1 lactic acid/glycolic acid ratio. In anotherembodiment, the ratio is 60:40. In another embodiment, the ratio is70:30. In another embodiment, the ratio is 80:20. In another embodiment,the ratio is 90:10. In another embodiment, the ratio is 95:5. In anotherembodiment, the ratio is another ratio appropriate for an extended invivo release profile, as defined herein. In another embodiment, theratio is 50:50. The PLGA may be either a random or block copolymer. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the biodegradable polyester is selected from thegroup consisting of a polycaprolactone, a polyhydroxyalkanoate, apolypropylenefumarate, a polyorthoester, a polyanhydride, and apolyalkylcyanoacrylate, provided that the polyester contains a hydrogenbond acceptor moiety. In another embodiment, the biodegradable polyesteris a block copolymer containing a combination of any two monomersselected from the group consisting of a PLA, PGA, a PLGA,polycaprolactone, a polyhydroxyalkanoate, a polypropylenefumarate, apolyorthoester, a polyanhydride, and a polyalkylcyanoacrylate. Inanother embodiment, the biodegradable polyester is a random copolymercontaining a combination of any two of the monomers listed above. Eachpossibility represents a separate embodiment of the present invention.

The molecular weight (MW) of a biodegradable polyester of methods andcompositions of the present invention is, in another embodiment, betweenabout 10-40 KDa. In another embodiment, the MW is between about 5-50KDa. In another embodiment, the MW is between about 15-40 KDa. Inanother embodiment, the MW is between about 20-40 KDa. In anotherembodiment, the MW is between about 15-35 KDa. In another embodiment,the MW is between about 10-35 KDa. In another embodiment, the MW isbetween about 10-30 KDa. In another embodiment, a mixture of PLGApolymers of different MW is utilized. In another embodiment, thedifferent polymers both have a MW in one of the above ranges. Eachpossibility represents a separate embodiment of the present invention.

Antibiotics

The antibiotic of methods and compositions of the present invention is,in another embodiment, doxycycline. In another embodiment, theantibiotic is a hydrophobic tetracycline. Non-limiting examples ofhydrophobic tetracycline are 6-demethyl-6-deoxytetracycline, 6-methylenetetracycline, minocycline (also known as7-dimethylamino-6-demethyl-6-deoxytetracycline), and13-phenylmercapto-a-6-deoxy-tetracycline. In another embodiment, theantibiotic is selected from the group consisting of doxycycline,tetracycline, and minocycline. In another embodiment, the antibiotic isintegrated into the matrix composition.

In another embodiment, the antibiotic is selected from the groupconsisting of amoxicillin, amoxicillin/clavulanic acid, penicillin,metronidazole, clindamycine, chlortetracycline, demeclocycline,oxytetracycline, amikacin, gentamicine, kanamycin, neomycin, netilmicin,streptomycin, tobramycin, cefadroxil, cefazolin, cephalexin,cephalothin, cephapirin, cephradine, cefaclor, cefamandole,cefametazole, cefonicid, cefotetan, cefoxitine, cefpodoxime, cefprozil,cefuroxime, cefdinir, cefixime, cefoperazone, cefotaxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefepime, azithromycin,clarithromycin, dirithromycin, erythromycin, lincomycin, troleandomycin,bacampicillin, carbenicillin, cloxacillin, dicloxacillin, meticillin,mezlocillin, nafcillin, oxacillin, piperacillin, ticarcillin, cinoxacin,ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin,nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, sulfisoxazole,sulfacytine, sulfadiazine, sulfamethoxazole, sulfisoxazole, dapson,aztreonam, bacitracin, capreomycin, chloramphenicol, clofazimine,colistimethate, colistin, cycloserine, fosfomycin, furazolidone,methenamine, nitrofurantoin, pentamidine, rifabutin, rifampin,spectinomycin, trimethoprim, trimetrexate glucuronate, and vancomycin.

In another embodiment, the biologically active ingredient is anantiseptic drug such as chlorhexidine.

Each antibiotic represents a separate embodiment of the presentinvention.

NSAID's

Any suitable NSAID may be integrated into the matrix composition forsustained and/or controlled release. The NSAID of methods andcompositions of the present invention is, in one embodiment,flurbiprofen. In another embodiment, the NSAID is selected from thegroup consisting of ibuprofen and flurbiprofen. In another embodiment,the NSAID is selected from the group consisting of ibuprofen,flurbiprofen, aminosalicylate sodium, choline magnesium trisalicylate,choline salicylate, diclofenac, diflunisal, etodolac, fenoprofen,indomethacin, ketoprofen, ketolac tromethamine, magnesium salicylate,meclofenamate, mefenamic acid, nabumetone, naproxen, oxaprozin,oxyphenbutazone, piroxicam, salsalate, sulindac, tolmetin.

Each NSAID represents a separate embodiment of the present invention.

Steroids

In another embodiment, the active agent of methods and compositions ofthe present invention is a steroid. According to one embodiment thesteroid is a steroidal anti-inflammatory drug. Non limiting examples ofsteroidal anti-inflammatory drugs (SAIDs) to be used in the formulationsof the present invention include, but are not limited to,Corticosteroids such as: betamethasone, betamethasone valerate,cortisone, dexamethasone, dexamethasone 21-phosphate, fludrocortisone,flumethasone, fluocinonide, fluocinonide desonide, fluocinolone,fluocinolone acetonide, fluocortolone, halcinonide, halopredone,hydrocortisone, hydrocortisone 17-valerate, hydrocortisone 17-butyrate,hydrocortisone 21-acetate methylprednisolone, prednisolone, prednisolone21-phosphate, prednisone, triamcinolone, triamcinolone acetonide,cortodoxone, fluoracetonide, fludrocortisone, difluorsone diacetate,flurandrenolone acetonide, medrysone, amcinafel, amcinafide,betamethasone and its other esters, chloroprednisone, clorcortelone,descinolone, desonide, dichlorisone, difluprednate, flucloronide,flumethasone, flunisolide, flucortolone, fluoromethalone, fluperolone,fluprednisolone, meprednisone, methylmeprednisolone, paramethasone,cortisone acetate, hydrocortisone cyclopentylpropionate, cortodoxone,flucetonide, fludrocortisone acetate, flurandrenolone acetonide,medrysone, amcinafal, amcinafide, betamethasone, betamethasone benzoate,chloroprednisone acetate, clocortolone acetate, descinolone acetonide,desoximetasone, dichlorisone acetate, difluprednate, flucloronide,flumethasone pivalate, flunisolide acetate, fluperolone acetate,fluprednisolone valerate, paramethasone acetate, prednisolamate,prednival, triamcinolone hexacetonide, cortivazol, formocortal andnivazol.

Anti-Cancer Agents

As referred to herein, the term “anti-cancer agent” refers to any typeof agent that may be used in the treatment of cancer and/or cancerrelated conditions. The anti-cancer reagent may include any naturallyoccurring or synthetically produced molecule that is capable ofaffecting directly or indirectly the growth and/or viability of cancercells, cancer tumor, and/or cancer related conditions and symptoms. Theanti-cancer agent may include, for example, a naturally occurringprotein or peptide, a modified protein or peptide, a recombinantprotein, a chemically synthesized protein or peptide, a low oralbioavailability protein or peptide, a chemical molecule, a syntheticchemical molecule, a chemotherapeutic drug, a biologically therapeuticdrug, and the like, or any combination thereof. The anti-cancer reagentmay be cytotoxic (toxic to cells) and/or cytostatic (suppress cellgrowth) and/or antiproliferative to the cancer cells and may exert itseffect on cancer cells directly and/or indirectly. According to someembodiments, the anti-cancer reagent may be administered alone and/or incombination and/or before and/or after one or more additional cancertreatments. The additional cancer treatment may include such treatmentsas, but not limited to: chemotherapy (use of drugs to affect the cancercells), radiotherapy (use of high-energy radiation of various sources toaffect the cancer cells); biological therapy (a therapy which helps theimmune system fight cancer); surgical procedures (surgical removal ofthe cancerous tumor); gene therapy; bone marrow transplantation; anyother therapy known in the art, or any combination thereof.

Non limiting examples of anti-cancer reagents and chemotherapeutic drugsmay include such drugs as, but not limited to: Alkaloids, such as, butnot limited to: Docetaxel, Etoposide, Irinotecan, Paclitaxel,Teniposide, Topotecan, Vinblastine, Vincristine, Vindesine; Alkylatingagents, such as, but not limited to: Busulfan, Improsulfan, Piposulfan,Benzodepa, Carboquone, Meturedepa, Uredepa, Altretamine,triethylenemelamine, Triethylenephosphoramide,Triethylenethiophosphoramide, Chlorambucil, Chloranaphazine,Cyclophosphamide, Estramustine, Ifosfamide, Mechlorethamine,Mechlorethamine Oxide Hcl, Melphalan, Novemebichin, PerfosfamidePhenesterine, Prednimustine, Trofosfamide, Uracil Mustard, Carmustine,Chlorozotocin, Fotemustine, Lomustine, Nimustine, Semustine Ranimustine,Dacarbazine, Mannomustine, Mitobronitol, Mitolactol, Pipobroman,Temozolomide; Antibiotics and analogs, such as, but not limited to:Aclacinomycins, Actinomycins, Anthramycin, Azaserine, Bleomycins,Cactinomycin, Carubicin, Carzinophilin, Cromomycins, Dactinomycins,Daunorubicin, 6-Diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin,Idarubicin, Menogaril, Mitomycins, Mycophenolic Acid, Nogalamycine,Olivomycins, Peplomycin, Pirarubicin, Plicamycin, Porfiromycin,Puromycine, Streptonigrin, Streptozocin, Tubercidin, Zinostatin,Zorubicin; Antimetabolites, such as, but not limited to: Denopterin,Edatrexate, Methotrexate, Piritrexim, Pteropterin, Tomudex,Trimetrexate, Cladridine, Fludarabine, 6-Mercaptopurine, PentostatineThiamiprine, Thioguanine, Ancitabine, Azacitidine, 6-Azauridine,Carmofur, Cytarabine, Doxifluridine, Emitefur, Floxuridine,Fluorouracil, Gemcitabine, Tegafur; Platinum complexes, such as, but notlimited to: Caroplatin, Cisplatin, Miboplatin, Oxaliplatin; alkylatorsincluding, but not limited to, busulfan (Myleran, Busulfex),chlorambucil (Leukeran), ifosfamide (with or without MESNA),cyclophosphamide (Cytoxan, Neosar), glufosfamide, melphalan, L-PAM(Alkeran), dacarbazine (DTIC-Dome), and temozolamide (Temodar);anthracyclines, including, but not limited to doxorubicin (Adriamycin,Doxil, Rubex), mitoxantrone (Novantrone), idarubicin (Idamycin),valrubicin (Valstar), and epirubicin (Ellence); antibiotics, including,but not limited to, dactinomycin, actinomycin D (Cosmegen), bleomycin(Blenoxane), daunorubicin, and daunomycin (Cerubidine, DanuoXome);aromatase inhibitors, including, but not limited to anastrozole(Arimidex) and letroazole (Femara); bisphosphonates, including, but notlimited to zoledronate (Zometa); cyclo-oxygenase inhibitors, including,but not limited to, celecoxib (Celebrex); estrogen receptor modulatorsincluding, but not limited to tamoxifen (Nolvadex) and fulvestrant(Faslodex); folate antagonists including, but not limited tomethotrexate and tremetrexate; inorganic aresenatesincluding, but notlimited to arsenic trioxide (Trisenox); microtubule inhibitors (e.g.taxanes) including, but not limited to vincristine (Oncovin),vinblastine (Velban), paclitaxel (Taxol, Paxene), vinorelbine(Navelbine), epothilone B or D or a derivative of either, anddiscodermolide or its derivatives, nitrosoureas including, but notlimited to procarbazine (Matulane), lomustine, CCNU (CeeBU), carmustine(BCNU, BiCNU, Gliadel Wafer), and estramustine (Emcyt); nucleosideanalogs including, but not limited to mercaptopurine, 6-MP (Purinethol),fluorouracil, 5-FU (Adrucil), thioguanine, 6-TG (Thioguanine),hydroxyurea (Hydrea), cytarabine (Cytosar-U, DepoCyt), floxuridine(FUDR), fludarabine (Fludara), pentostatin (Nipent), cladribine(Leustatin, 2-CdA), gemcitabine (Gemzar), and capecitabine (Xeloda);osteoclast inhibitors including, but not limited to pamidronate(Aredia); platinum containing compounds including, but not limited tocisplatin (Platinol) and carboplatin (Paraplatin); retinoids including,but not limited to tretinoin, ATRA (Vesanoid), alitretinoin (Panretin),and bexarotene (Targretin); topoisomerase 1 inhibitors including, butnot limited to topotecan (Hycamtin) and irinotecan (Camptostar);topoisomerase 2 inhibitors including, but not limited to etoposide,VP-16 (Vepesid), teniposide, VM-26 (Vumon), and etoposide phosphate(Etopophos); tyrosine kinase inhibitors including, but not limited toimatinib (Gleevec); various other proteins including monoclonalantibodies, peptides and enzymes, various other molecules, such as, forexample, Super Oxide dismutase (SOD), leptin; flavanoids; or anycombinations thereof.

Non limiting examples of anti-cancer agents and biological therapiesthat may be used according to some embodiments, may include, suchtherapies and molecules as, but not limited to: administration of animmunomodulatory molecule, such as, for example, a molecule selectedfrom the group consisting of tumor antigens, antibodies, cytokines (suchas, for example, interleukins (such as, for example, interleukin 2,interleukin 4, interleukin 12), interferons (such as, for example,interferon El interferon D, interferon alpha), tumor necrosis factor(TNF), granulocyte macrophage colony stimulating factor (GM-CSF),macrophage colony stimulating factor (M-CSF), and granulocyte colonystimulating factor (G-CSF)), tumor suppressor genes, chemokines,complement components, complement component receptors, immune systemaccessory molecules, adhesion molecules, adhesion molecule receptors,agents affecting cell bioenergetics, or any combinations thereof.

Osteogenic Factors

In another embodiment, the active agent of methods and compositions ofthe present invention is a compound which induces or stimulates theformation of bone. In another embodiment the active agent is osteogenicfactor. In another embodiment, the osteogenic factor refers to anypeptide, polypeptide, protein or any other compound or composition whichinduces or stimulates the formation of bone. In another embodiment, theosteogenic factor induces differentiation of bone repair cells into bonecells, such as osteoblasts or osteocytes. In another embodiment theosteogenic factor is selected from the group consisting of TGF-beta, BMPand FGF. In another embodiment the osteogenic factor is encapsulatedwithin the matrix composition of the present invention in aconcentration sufficient to induce differentiation of bone repair cellsinto bone cells which form bone.

Bone Resorption Inhibitors

In another embodiment, the active agent of methods and compositions ofthe present invention is a compound useful for supporting bone recovery.In another embodiment, the active agent is a bone resorption inhibitor.In another embodiment, the active agent is a bone density conservationagent. In another embodiment, the compound is selected from the groupconsisting of osteoprotegerin (OPG), BMP-2, BMP-4, vascular endothelialgrowth factor (VEGF), alendronate, etidronate disodium, pamidronate,risedronate, and tiludronate. In another embodiment, the compound isosteoprotegerin (OPG), a naturally secreted decoy receptor that inhibitsosteoclast maturation and activity and induces osteoclast apoptosis. Inanother embodiment, the active agent is a bone restructuring element.Non-limiting examples of bone restructuring elements are BMP peptides.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the compound is a bone morphogenetic protein(BMP). In another embodiment, the compound is selected from the groupconsisting of BMP-2 and BMP-4, which accelerate osteoblast activity.

In another embodiment, the compound is vascular endothelial growthfactor (VEGF).

In another embodiment, the compound is an estrogen. In anotherembodiment, the compound is selected from the group consisting ofbisphosphonate derivative. In another embodiment, the bisphosphonatederivative is selected from the group consisting of alendronate,etidronate disodium, pamidronate, risedronate, and tiludronate.

Each compound represents a separate embodiment of the present invention.

Anti-Fungal Agents

In another embodiment, the biologically active ingredient is anantifungal drug, e.g. amphotericin B cholesteryl sulfate complex,natamycin, amphotericine, clotrimazole, nystatin, amphotericin B lipidcomplex, fluconazole, flucytosine, griseofulvin, itraconazole,ketoconazole, benzoic acid and salicylic acid, betamethasone andclotrimazole, butenafine, carbol-fuchsin, ciclopirox, clioquinol,clioquinol and hydrocortisone, clotrimazole, econazole, gentian violet,haloprogin, iodoquinol and hydrocortisone, ketoconazole, miconazole,naftifine, nystatin, nystatin and triamcinolone, oxiconazole, sodiumthiosulfate, sulconazole, terbinafine, tolnaftate, triacetin,undecylenic acid and derivatives thereof, butoconazole, clotrimazole,sulfanilamide, terconazole, and tioconazole.

Targeting Moieties

In another embodiment, a matrix composition of methods and compositionsof the present invention further comprises a targeting moiety capable ofinteracting with a target molecule. Preferably the target molecule isselected from the group consisting of a collagen molecule, a fibrinmolecule and a heparin. In another embodiment, the target molecule isanother surface molecule that forms part of the extracellular matrix(ECM) of a target cell. ECM is produced and assembled locally by cells.The most important cells involved in assembling and maintaining ECM arefibroblasts. ECM contains polysaccharide chains called GAGs(glyosaminoglycans) and various protein fibers e.g., collagen, elastin,fibronectin and laminin.

In another embodiment, the targeting moiety is a fibronectin peptide.Fibronectin is a high-molecular-weight glycoprotein that binds ECMcomponents such as collagen, fibrin and heparin. In another embodiment,the targeting moiety is another targeting moiety capable of interactionwith a target molecule selected from the group consisting of a collagenmolecule, a fibrin molecule and a heparin. Each possibility represents aseparate embodiment of the present invention.

“Fibronectin peptide” refers, in another embodiment, to a full-lengthfibronectin protein. In another embodiment, the term refers to afragment of fibronectin. In another embodiment, the fragment includesthe collagen binding domain. Collagen binding domains of fibronectinmolecules are well known in the art, and are described, for example, inHynes, R O (1990). Fibronectins. New York: Springer-Verlag and inYamada, K M and Clark, R A F (1996). Provisional matrix. In TheMolecular and Cellular Biology of Wound Repair (ed. R. A. F. Clark), pp.51-93. New York: Plenum Press. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the targeting moiety is incorporated into thematrix composition. In another embodiment, the targeting moiety ismodified to confer ability to incorporate into the lipid matrix. Inanother embodiment, the modification comprises binding to a lipidmoiety. A non-limiting example of a lipid moiety is hydrogenatedphosphatidylethanolamine (HPE). However, any lipid moiety capable ofincorporation into the lipid matrix is suitable. In another embodiment,the targeting moiety is able to be incorporated into the lipid matrixwithout modification. In another embodiment, the targeting moiety isattached to the surface of a matrix composition of the presentinvention. In another embodiment, the targeting moiety is bound to thesurface of the matrix composition or vesicle by a hydrophobic anchorcovalently bound to the targeting moiety. In another embodiment, thetargeting moiety is bound to the lipid vesicles by a hydrophobic anchor.In another embodiment, the targeting moiety is included during thepreparation of the drug carrier, allowing it to be located in deeperlayers of the carrier. Each possibility represents a separate embodimentof the present invention.

In one embodiment, the target molecule is a collagen. Collagens are wellknown in the art, and are described, for example, in Khoshnoodi J et al(Molecular recognition in the assembly of collagens: terminalnoncollagenous domains are key recognition modules in the formation oftriple helical protomers. J Biol. Chem. 281(50):38117-21, 2006). Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the target molecule is a fibrin. Fibrins are wellknown in the art, and are described, for example, in Valenick L V et al(Fibronectin fragmentation promotes alpha4beta1 integrin-mediatedcontraction of a fibrin-fibronectin provisional matrix. Exp Cell Res309(1):48-55, 2005) and Mosesson MW (Fibrinogen and fibrin structure andfunctions. J Thromb Haemost 3(8):1894-904, 2005). Each possibilityrepresents a separate embodiment of the present invention.

In one embodiment, the target molecule is a heparin. Heparins are wellknown in the art, and are described, for example, in Mosesson MW(Fibrinogen and fibrin structure and functions. J Thromb Haemost3(8):1894-904, 2005). Each possibility represents a separate embodimentof the present invention.

Additional Components

In another embodiment, a matrix composition of methods and compositionsof the present invention further comprises a free fatty acid. In anotherembodiment, the free fatty acid is an omega-6 fatty acid. In anotherembodiment, the free fatty acid is an omega-9 fatty acid. In anotherembodiment, the free fatty acid is selected from the group consisting ofomega-6 and omega-9 fatty acids. In another embodiment, the free fattyacid has 14 or more carbon atoms. In another embodiment, the free fattyacid has 16 or more carbon atoms. In another embodiment, the free fattyacid has 16 carbon atoms. In another embodiment, the free fatty acid has18 carbon atoms. In another embodiment, the free fatty acid has 16-22carbon atoms. In another embodiment, the free fatty acid has 16-20carbon atoms. In another embodiment, the free fatty acid has 16-18carbon atoms. In another embodiment, the free fatty acid has 18-22carbon atoms. In another embodiment, the free fatty acid has 18-20carbon atoms. In another embodiment, the free fatty acid is linoleicacid. In another embodiment, the free fatty acid is linolenic acid. Inanother embodiment, the free fatty acid is oleic acid. In anotherembodiment, the free fatty acid is selected from the group consisting oflinoleic acid, linolenic acid, and oleic acid. In another embodiment,the free fatty acid is another appropriate free fatty acid known in theart. In another embodiment, the free fatty acid adds flexibility to thematrix composition. In another embodiment, the free fatty acid slows thein vivo release rate. In another embodiment, the free fatty acidimproves the consistency of the in vivo controlled release. In someembodiments the fatty acid is unsaturated. In another embodiment, thefree fatty acid is saturated. In another embodiment, incorporation of asaturated fatty acid having at least 14 carbon atoms increases thegel-fluid transition temperature of the resulting matrix composition.

In another embodiment, the free fatty acid is deuterated. In anotherembodiment, deuteration of the lipid acyl chains lowers the gel-fluidtransition temperature.

In another embodiment, a free fatty acid is incorporated into the matrixcomposition. Each type of fatty acid represents a separate embodiment ofthe present invention.

In another embodiment, a matrix composition of methods and compositionsof the present invention further comprises a tocopherol. The tocopherolof methods and compositions of the present invention is, in anotherembodiment, E307 (α-tocopherol). In another embodiment, the tocopherolis β-tocopherol. In another embodiment, the tocopherol is E308(γ-tocopherol). In another embodiment, the tocopherol is E309(δ-tocopherol). In another embodiment, the tocopherol is selected fromthe group consisting of α-tocopherol, β-tocopherol, γ-tocopherol, andδ-tocopherol. In another embodiment, the tocopherol is incorporated intothe matrix composition. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a matrix composition of methods and compositionsof the present invention further comprises physiologically acceptablebuffer salts, which are well known in the art. Non-limiting examples ofphysiologically acceptable buffer salts are phosphate buffers. A typicalexample of a phosphate buffer is 40 parts NaCl, 1 part KCl, 7 partsNa₂HPO₄.2H₂O and 1 part KH₂PO₄. In another embodiment, the buffer saltis any other physiologically acceptable buffer salt known in the art.Each possibility represents a separate embodiment of the presentinvention.

Release Rates and General Characteristics of the Matrix Compositions

The in vivo release time of 90% of the active ingredient for matrixcompositions of the present invention is preferably between 1 week and 6months. In another embodiment, the release time is between 4 days and 6months. In another embodiment, the release time is between 1 week and 5months. In another embodiment, the release time is between 1 week and 5months. In another embodiment, the release time is between 1 week and 4months. In another embodiment, the release time is between 1 week and 3months. In another embodiment, the release time is between 1 week and 2months. In another embodiment, the release time is between 2 weeks and 6months. In another embodiment, the release time is between 2 weeks and 5months. In another embodiment, the release time is between 2 weeks and 4months. In another embodiment, the release time is between 2 weeks and 3months. In another embodiment, the release time is between 3 weeks and 6months. In another embodiment, the release time is between 3 weeks and 5months. In another embodiment, the release time is between 3 weeks and 4months. In another embodiment, the release time is between 3 weeks and 3months. Each possibility represents a separate embodiment of the presentinvention.

Methods for modulating the release rate of biodegradable polymerimplants (in the absence of lipids) and drug-containing vesicles (in theabsence of biodegradable polymer) are well known in the art. Forexample, in the case of polymers, increasing the lactic acid:co-glycolicacid ratio of PLGA will extend the release time. In the case ofdrug-containing vesicles, increasing the amount of cholesterol willextend the release time. Each of these methods can be used to modulatethe release rate of the matrix compositions of the present invention.

“Biodegradable,” as used herein, refers to a substance capable of beingdecomposed by natural biological processes at physiological pH.“Physiological pH” refers to the pH of body tissue, typically between6-8. “Physiological pH” does not refer to the highly acidic pH ofgastric juices, which is typically between 1 and 3.

The weight ratio of total lipids to the polymer in order to achievelipid saturation can be determined by a number of methods, as describedherein. In another embodiment, the lipid:polymer weight ratio of acomposition of the present invention is between 1:1 and 9:1 inclusive.In another embodiment, the ratio is between 2:1 and 9:1 inclusive. Inanother embodiment, the ratio is between 3:1 and 9:1 inclusive. Inanother embodiment, the ratio is between 4:1 and 9:1 inclusive. Inanother embodiment, the ratio is between 5:1 and 9:1 inclusive. Inanother embodiment, the ratio is between 6:1 and 9:1 inclusive. Inanother embodiment, the ratio is between 7:1 and 9:1 inclusive. Inanother embodiment, the ratio is between 8:1 and 9:1 inclusive. Inanother embodiment, the ratio is between 1.5:1 and 9:1 inclusive. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment for purposes of illustration, in the case whereinthe polymer is predominantly 40 KDa PLGA (poly (lactic-co-glycolic acid,1:1 ratio)), the molar ratio of total lipids to 40 KDa PLGA is typicallyin the range of 20-100 inclusive. In another embodiment, the molar ratioof total lipids to 40 KDa PLGA is between 20-200 inclusive. In anotherembodiment, the molar ratio is between 10-100 inclusive. In anotherembodiment, the molar ratio is between 10-200 inclusive. In anotherembodiment, the molar ratio is between 10-50 inclusive. In anotherembodiment, the molar ratio is between 20-50 inclusive. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the melting temperature (T_(m)) of a matrixcomposition of the present invention is at least 37° C. In anotherembodiment, the T_(n), is at least 40° C. In another embodiment, theT_(m) is at least 42° C. In another embodiment, the T_(m) is at least44° C. In another embodiment, the T_(m) is at least 46° C. In anotherembodiment, the T_(m) is at least 48° C. In another embodiment, theT_(m) is at least 50° C. Each possibility represents a separateembodiment of the present invention.

Implants and Other Pharmaceutical Compositions

In another embodiment, a matrix composition of the present invention isin the form of an implant, following evaporation of the organicsolvents. The evaporation of the solvents is typically done attemperatures ranging from 20 to 60° C.

In another embodiment, the implant is homogeneous. In anotherembodiment, the implant is manufactured by a process comprising the stepof freeze-drying the material in a mold. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides an implantcomprising an antibiotic-containing matrix composition of the presentinvention. In another embodiment, the present invention provides animplant comprising an NSAID-containing matrix composition of the presentinvention. In another embodiment, the present invention provides animplant comprising a bone resorption inhibitor-containing matrixcomposition of the present invention. In another embodiment, the presentinvention provides an implant comprising a matrix composition of thepresent invention that contains an antibiotic and an NSAID. In anotherembodiment, the present invention provides an implant comprising amatrix composition of the present invention that contains an antibioticand a bone resorption inhibitor. In another embodiment, the presentinvention provides an implant comprising a matrix composition of thepresent invention that contains a bone resorption inhibitor and anNSAID. In another embodiment, the present invention provides an implantcomprising a matrix composition of the present invention that containsan antibiotic, an NSAID, and a bone resorption inhibitor. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the process of creating an implant from acomposition of the present invention comprises the steps of (a) creatinga matrix composition according to a method of the present invention inthe form of a bulk material; (b) transferring the bulk material into amold or solid receptacle of a desired shaped; (c) freezing the bulkmaterial; and (d) lyophilizing the bulk material.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising a matrix composition of the present invention anda pharmaceutically acceptable excipient.

In another embodiment, a matrix composition of the present invention isin the form of microspheres, following evaporation of the organicsolvents. In another embodiment, the microspheres are homogeneous. Inanother embodiment, the microspheres are manufactured by a processcomprising the step of spray-drying. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides microspheres madeof a matrix composition of the present invention. In another embodiment,the present invention provides a pharmaceutical composition comprisingmicrospheres of the present invention and a pharmaceutically acceptableexcipient. In another embodiment, the pharmaceutical composition is in aparenterally injectable form. In another embodiment, the pharmaceuticalcomposition is in an infusible form. In another embodiment, theexcipient is compatible for injection. In another embodiment, theexcipient is compatible for infusion. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the particle size of microspheres of the presentinvention is approximately 500-2000 nm. In another embodiment, theparticle size is about 400-2500 nm. In another embodiment, the particlesize is about 600-1900 nm. In another embodiment, the particle size isabout 700-1800 nm. In another embodiment, the particle size is about500-1800 nm. In another embodiment, the particle size is about 500-1600nm. In another embodiment, the particle size is about 600-2000 nm. Inanother embodiment, the particle size is about 700-2000 nm. In anotherembodiment, the particles are of any other size suitable forpharmaceutical administration. Each possibility represents a separateembodiment of the present invention.

Therapeutic Methods

In another embodiment, the present invention provides a method ofadministering an antibiotic to a subject in need thereof, the methodcomprising the step of administering to the subject a matrix compositionof the present invention, thereby administering an antibiotic to asubject in need thereof. In another embodiment, a pharmaceuticalcomposition comprising the matrix composition is administered. Inanother embodiment, an implant comprising the matrix composition isadministered. In another embodiment, an injectable formulationcomprising the matrix composition is injected. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofadministering a non-steroidal anti-inflammatory drug (NSAID) to asubject in need thereof, the method comprising the step of administeringto the subject a matrix composition of the present invention, therebyadministering an NSAID to a subject in need thereof. In anotherembodiment, a pharmaceutical composition comprising the matrixcomposition is administered. In another embodiment, an implantcomprising the matrix composition is administered. In anotherembodiment, an injectable formulation comprising the matrix compositionis injected. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a pharmaceuticalcomposition for administering an antibiotic to a subject in needthereof, comprising a matrix composition of the present invention. Inanother embodiment, the pharmaceutical composition is an implant. Inanother embodiment, the pharmaceutical composition is an injectablecomposition. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a pharmaceuticalcomposition for administering an NSAID to a subject in need thereof,comprising a matrix composition of the present invention. In anotherembodiment, the pharmaceutical composition is an implant. In anotherembodiment, the pharmaceutical composition is an injectable composition.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a pharmaceuticalcomposition for co-administering an antibiotic and an NSAID to a subjectin need thereof, comprising a matrix composition of the presentinvention. In another embodiment, the pharmaceutical composition is animplant. In another embodiment, the pharmaceutical composition is aninjectable composition. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method oftreating periodontitis in a subject in need thereof, said methodcomprising the step of administering to said subject a matrixcomposition of the present invention, thereby treating periodontitis.“Periodontitis” refers to an inflammatory diseases affecting the tissuesthat surround and support the teeth. In another embodiment,periodontitis involves progressive loss of the alveolar bone around theteeth and may eventually lead to the loosening and subsequent loss ofteeth if left untreated. Periodontitis in some cases has a bacterialetiology. In another embodiment, the periodontitis is a chronicperiodontitis. In another embodiment, the periodontitis is any othertype of periodontitis known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides a method ofstimulating bone augmentation in a subject in need thereof, said methodcomprising the step of administering to said subject a matrixcomposition of the present invention, thereby stimulating boneaugmentation. In another embodiment, the subject has a disease ordisorder selected from the group consisting of osteosarcoma/malignantfibrous histiocytoma of bone (PDQ), osteosarcoma, chondrosarcoma,Ewing's sarcoma, malignant fibrous histiocytoma, fibrosarcoma andmalignant fibrous histiocytoma, giant cell tumor of bone, chordoma,lymphoma, multiple myeloma, osteoarthritis, Paget's disease of bone,arthritis, degenerative changes, osteoporosis, osteogenesis imperfecta,bone spurs, renal osteodystrophy, hyperparathyroidism, osteomyelitis,enchondroma, osteochondroma, osteopetrosis, and a diabetes-associatedbone or joint disorder. In another embodiment, the matrix composition isin the form of an implant. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method ofreducing an incidence of complications from orthopedic surgery in asubject in need thereof, said method comprising the step ofadministering to said subject a matrix composition of the presentinvention, thereby reducing an incidence of complications fromorthopedic surgery. In another embodiment, the orthopedic surgery isselected from the group consisting of hand surgery, shoulder and elbowsurgery, total joint reconstruction (arthroplasty), pediatricorthopedics, foot and ankle surgery, spine surgery, knee arthroscopy,knee meniscectomy, shoulder arthroscopy, shoulder decompression, carpaltunnel release, knee chondroplasty, removal of a support implant, kneeanterior cruciate ligament reconstruction, knee replacement, repair offemoral neck fracture, repair of trochanteric fracture, debridement ofskin, muscle, or bone fracture, repair of knee menisci, hip replacement,shoulder arthroscopy/distal clavicle excision, repair of rotator cufftendon, repair fracture of radius (bone)/ulna, laminectomy, repair ofankle fracture (bimalleolar type), shoulder arthroscopy and debridement,lumbar spinal fusion, repair fracture of the distal part of radius, lowback intervertebral disc surgery, incise finger tendon sheath, repair ofankle fracture (fibula), repair of femoral shaft fracture, and repair oftrochanteric fracture. In another embodiment, the matrix composition isin the form of an implant. In another embodiment, the implant isadministered during the orthopedic surgery. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofenhancing an effectiveness of surgical regenerative procedure in asubject in need thereof, said method comprising the step ofadministering to said subject a matrix composition of the presentinvention, thereby enhancing an effectiveness of surgical regenerativeprocedure. In another embodiment, the surgical regenerative procedure isa periodontal procedure. In another embodiment, the surgicalregenerative procedure comprises administering an implant (animplantology procedure). In another embodiment, the implantologyprocedure is directed to ridge or sinus augmentation. In anotherembodiment, the matrix composition is in the form of an implant. Inanother embodiment, the implant is administered during the surgicalprocedure. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method oftreating an osteomyelitis in a subject in need thereof, said methodcomprising the step of administering to said subject a matrixcomposition of the present invention, thereby treating an osteomyelitis.In another embodiment, the matrix composition is in the form of animplant. In another embodiment, the implant is administered at or nearthe site of osteomyelitis. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a matrix composition of the present invention isadministered for aiding orthopedic bone and soft tissue recovery. Thecompounds are administered during or after a procedure selected from thegroup consisting of knee arthroscopy and meniscectomy, shoulderarthroscopy and decompression, carpal tunnel release, knee arthroscopyand chondroplasty, removal of support implant, knee arthroscopy andanterior cruciate ligament reconstruction, knee replacement, repair offemoral neck fracture, repair of trochanteric fracture, debridement ofskin/muscle/bone/fracture, knee arthroscopy repair of both menisci, hipreplacement, shoulder arthroscopy/distal clavicle excision, repair ofrotator cuff tendon, repair fracture of radius (bone)/ulna, laminectomy,repair of ankle fracture (bimalleolar type), shoulder arthroscopy anddebridement, lumbar spinal fusion, repair fracture of the distal part ofradius, low back intervertebral disc surgery, incise finger tendonsheath, repair of ankle fracture (fibula), repair of femoral shaftfracture, and repair of trochanteric fracture.

In another embodiment, a matrix composition of the present invention isadministered for homeostasis, reducing infections and avoiding tissueadhesions by the use of products such as sponges and membranes.

In another embodiment, a matrix composition of the present invention isadministered for reducing of inflammatory reaction around suturematerials.

Methods of Making Matrix Compositions

In order to obtain the compositions of the invention any suitable methodmay be employed that will yield a homogeneous dispersion of the polymerand the lipids in a water resistant matrix. Advantageously according tosome embodiments the methods employed eschew the use of water at anystage of the manufacturing process.

According to some embodiments the polymer is mixed separately withappropriate selected volatile organic solvent(s) on the one hand and thephospholipids together with the active pharmaceutical agent are mixedwith its appropriate selected solvent(s) or solvents prior to mixingtogether with the polymer.

In certain embodiments, the present invention provides a method ofproducing a matrix composition, the method comprising the steps of:

(a) mixing into a first volatile organic solvent: (i) a biodegradablepolyester and (ii) sterol; and

(b) mixing separately into a second volatile organic solvent: (i) anactive agent; (ii) a phosphatidylcholine and optionally (iii) aphosphatidylethanolamine; and

(c) mixing and homogenizing the products resulting from steps (a) and(b).

In another embodiment, phosphatidylethanolamine is included in thevolatile organic solvent of step (a) instead of or in addition to aphosphatidylethanolamine added to the volatile organic solvent of step(b). In another embodiment, the biodegradable polyester is selected fromthe group consisting of PLA, PGA and PLGA. In another embodiment, thebiodegradable polyester is any other suitable biodegradable polyesterknown in the art. In some embodiments the first volatile organic solventis a non-polar solvent. In some embodiments the second volatile organicsolvent is a water miscible solvent. In cases where the active agent isa protein or peptide it is important to select solvents that will notdenature or impair the activity of the protein. In particularembodiments the active agent is selected from the group consisting of anNSAID, an antibiotic, an antifungal agent, a steroid, an anticanceragent, an osteogenic factor and a bone resorption inhibitor and mixturesthereof.

In another embodiment, the mixture of step (a) containing a volatileorganic solvent is homogenized prior to mixing it with the solution ofstep (b). In another embodiment, the volatile organic solvent or mixtureof volatile organic solvents used in step (a) may be same or differentthan the volatile organic solvent or mixture of organic solvents used instep (b). In another embodiment, the mixture of step (b) is homogenizedprior to mixing it with the mixture of step (a). In another embodiment,the polymer in the mixture of step (a) is lipid saturated. In anotherembodiment, the matrix composition is lipid saturated. Preferably, thepolymer and the phosphatidylcholine are incorporated into the matrixcomposition. In another embodiment, the active agent as well isincorporated into the matrix composition. In another embodiment, thematrix composition is in the form of a lipid-saturated matrix whoseshape and boundaries are determined by the biodegradable polymer. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the phosphatidylethanolamine of methods andcompositions of the present invention has saturated fatty acid moieties.In another embodiment, the fatty acid moieties have at least 14 carbonatoms. In another embodiment, the fatty acid moieties have 14-18 carbonatoms. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the phosphatidylcholine of methods andcompositions of the present invention has saturated fatty acid moieties.In another embodiment, the fatty acid moieties have at least 14 carbonatoms. In another embodiment, the fatty acid moieties have at least 16carbon atoms. In another embodiment, the fatty acid moieties have 14-18carbon atoms. In another embodiment, the fatty acid moieties have 16-18carbon atoms. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the weight ratio of total lipids to polymer inthe first volatile organic solvent is such that the polymer in thismixture is lipid-saturated. In another embodiment for purposes ofillustration, in the case wherein the polymer is predominantly 50 KDaPLGA (poly (lactic-co-glycolic acid, 1:1 ratio)), the molar ratio oftotal lipids to 50 KDa PLGA is typically in the range of 10-50inclusive. In another embodiment, the molar ratio of total lipids to 50KDa PLGA is between 10-100 inclusive. In another embodiment, the molarratio is between 20-200 inclusive. In another embodiment, the molarratio is between 20-300 inclusive. In another embodiment, the molarratio is between 30-400 inclusive. Each possibility represents aseparate embodiment of the present invention.

Each of the components of the above method and other methods of thepresent invention is defined in the same manner as the correspondingcomponent of the matrix compositions of the present invention.

In another embodiment, step (a) of the production method furthercomprises adding to the volatile organic solvent aphosphatidylethanolamine. In another embodiment, thephosphatidylethanolamine is the same phosphatidylethanolamine includedin step (b). In another embodiment, the phosphatidylethanolamine is adifferent phosphatidylethanolamine that may be any otherphosphatidylethanolamine known in the art. In another embodiment, thephosphatidylethanolamine is selected from the group consisting of thephosphatidylethanolamine of step (b) and a differentphosphatidylethanolamine. Each possibility represents a separateembodiment of the present invention.

In another embodiment, step (a) of the production method furthercomprises adding to the volatile organic solvent a tocopherol.

In another embodiment, step (b) of the production method furthercomprises adding to the volatile organic solvent physiologicallyacceptable buffer salts. Non-limiting examples of physiologicallyacceptable buffer salts are phosphate buffers. A typical example of aphosphate buffer is 40 parts NaCl, 1 part KCl, 7 parts Na₂HPO₄.2H₂O and1 part KH₂PO₄. In another embodiment, the buffer salt is any otherphysiologically acceptable buffer salt known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, step (b) of the production method furthercomprises adding to the volatile organic solvent a phospholipid selectedfrom the group consisting of a phosphatidylserine, aphosphatidylglycerol, a sphingomyelin, and a phosphatidylinositol.

In another embodiment, step (b) of the production method furthercomprises adding to the volatile organic solvent a sphingolipid. Inanother embodiment, the sphingolipid is ceramide. In another embodiment,the sphingolipid is a sphingomyelin. In another embodiment, thesphingolipid is any other sphingolipid known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, step (b) of the production method furthercomprises adding to the water-miscible, volatile organic solvent anomega-6 or omega-9 free fatty acid. In another embodiment, the freefatty acid has 16 or more carbon atoms. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, each step of the production method issubstantially free of aqueous solution. In another embodiment, each stepis substantially free of the presence of water or any aqueous solution.As provided herein, producing matrix compositions of the presentinvention in a water-free process enables lipid saturation.

Upon mixing, a homogenous mixture is formed, since the polymer islipid-saturated in the mixture of step (a). In another embodiment, thehomogenous mixture takes the form of a homogenous liquid. In anotherembodiment, upon freeze-drying or spray-drying the mixture, vesicles areformed. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the production method further comprises the stepof evaporating the solvent present in the product of step (c). Inanother embodiment, the evaporation utilizes atomization of the mixture.In another embodiment, the mixture is atomized into dry, heated air.Typically, atomization into heated air evaporates all water immediately,obviating the need for a subsequent drying step. In another embodiment,the mixture is atomized into a water-free solvent. In anotherembodiment, the evaporation is performed by spray drying. In anotherembodiment, the evaporation is performed by freeze drying. In anotherembodiment, the evaporation is performed using liquid nitrogen. Inanother embodiment, the evaporation is performed using liquid nitrogenthat has been pre-mixed with ethanol. In another embodiment, theevaporation is performed using another suitable technique known in theart. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a method of the present invention furthercomprises the step of vacuum-drying the composition. In anotherembodiment, the step of vacuum-drying is performed following the step ofevaporating. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the method of the present invention furthercomprises the step of evaporating the organic volatile solvent byheating the product of step (c). The heating is continuing until thesolvent is eliminated and in a typical temperature between roomtemperature to 60° C. In another embodiment a step of vacuum-drying isperformed following the step of solvent evaporation. Each possibilityrepresents a separate embodiment of the present invention.

Lipid Saturation and Techniques for Determining Same

“Lipid saturated,” as used herein, refers to saturation of the polymerof the matrix composition with phospholipids in combination with anyhydrophobic drug and targeting moiety present in the matrix, and anyother lipids that may be present. As described herein, matrixcompositions of the present invention comprise, in some embodiments,phospholipids other than phosphatidylcholine. In other embodiments, thematrix compositions comprise lipids other than phospholipids. The matrixcomposition is saturated by whatever lipids are present. “Saturation”refers to a state wherein the matrix contains the maximum amount oflipids of the type utilized that can be incorporated into the matrix.Methods for determining the polymer:lipid ratio to attain lipidsaturation and methods of determining the degree of lipid saturation ofa matrix are described herein. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the matrix composition of methods andcompositions of the present invention is substantially free of water.“Substantially free of water” refers, in another embodiment, to acomposition containing less than 1% water by weight. In anotherembodiment, the term refers to a composition containing less than 0.8%water by weight. In another embodiment, the term refers to a compositioncontaining less than 0.6% water by weight. In another embodiment, theterm refers to a composition containing less than 0.4% water by weight.In another embodiment, the term refers to a composition containing lessthan 0.2% water by weight. In another embodiment, the term refers to theabsence of amounts of water that affect the water-resistant propertiesof the composition. In another embodiment, the term refers to acomposition manufactured without the use of any aqueous solvents. Inanother embodiment, producing the composition using a processsubstantially free of water, as described herein, enables lipidsaturation. Lipid saturation confers upon the matrix composition abilityto resist bulk degradation in vivo; thus, the matrix compositionexhibits the ability to mediate extended release on a scale of severalweeks or months. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the matrix composition is essentially free ofwater. “Essentially free” refers to a composition comprising less than0.1% water by weight. In another embodiment, the term refers to acomposition comprising less than 0.08% water by weight. In anotherembodiment, the term refers to a composition comprising less than 0.06%water by weight. In another embodiment, the term refers to a compositioncomprising less than 0.04% water by weight. In another embodiment, theterm refers to a composition comprising less than 0.02% water by weight.In another embodiment, the term refers to a composition comprising lessthan 0.01% water by weight. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the matrix composition is free of water. Inanother embodiment, the term refers to a composition not containingdetectable amounts of water. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the matrix composition is dry. “Dry” refers, inanother embodiment, to the absence of detectable amounts of water ororganic solvent.

In another embodiment, the water permeability of the matrix compositionhas been minimized. “Minimizing” the water permeability refers to aprocess of producing the matrix composition in organic solvents, asdescribed herein, in the presence of an the amount of lipid that hasbeen determined to minimize the permeability to penetration of addedwater. The amount of lipid required can be determined by hydrating thevesicles with a solution containing tritium-tagged water, as describedherein.

In another embodiment, “lipid saturation” refers to filling of internalgaps (free volume) within the lipid matrix as defined by the externalborder of the polymeric backbone. The gaps are filled with thephospholipids in combination with other type of lipids, hydrophobic drugand targeting moiety present in the matrix, to the extent thatadditional lipid moieties can no longer be incorporated into the matrixto an appreciable extent.

In one embodiment, the following method is used to determine the degreeof lipid saturation:

Following manufacture, vesicles are hydrated and isolated bycentrifugation or filtration. Lipids that not entrapped in the vesiclesform free micelles or liposomes and are located in the supernatant. Theoverall lipid contents of the supernatant and the vesicles arequantified. In this manner, the entrapped vs. free lipid contents aredetermined for various formulation containing different lipid:polymerratios at the outset. Thus, the actual, experimental, maximumlipid/polymer ratio is determined.

In another embodiment, the following method is used to determine thedegree of lipid saturation:

Following manufacture, vesicles are hydrated with a solution containingtritium-tagged water, washed with tritium-free solution, and isolated bycentrifugation or filtration, and the amount of water entrapped perpolymer mass is quantified. This is repeated with differentlipid:polymer ratios, in order to determine the amount of lipidsrequired to saturate the free volume in the polymeric vesicles.

“Zero-order release rate” or “zero order release kinetics” means aconstant, linear, continuous, sustained and controlled release rate ofthe pharmaceutical active agent from the polymer matrix, i.e. the plotof amounts of pharmaceutical active agent released vs. time is linear.

EXPERIMENTAL DETAILS SECTION

Abbreviations used: phosphoethanolamine=PE; phosphatidylcholine=PC;1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine=DMPE (14:0);1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine=DPPE (16:0);1,2-distearoyl-sn-glycero-3-phosphocholine=DSPC (18:0);1,2-dipalmitoyl-sn-glycero-3-phosphocholine=DPPC (16:0);1,2-dioleoyl-sn-glycero-3-phosphocholine=DOPC (18:1);1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine=NBD-PC

Example 1 Platform Technology for Production of Drug CarrierCompositions Overview

To produce lipid-saturated polymer matrices, two mixtures are created.

1. A biodegradable polymer and a sterol and/or phospholipid componentare mixed with a volatile organic solvent, which is mixed to yield asolution or suspension of lipid-saturated polymer matrix, as measured byits differential scanning calorimetric (DSC) profile.2. The active agent and a phospholipid component are mixed with a secondvolatile organic solvent to yield a second solution or suspension.3. The two solutions or suspensions are combined and mixed untilequilibrium is reached; the organic solvents are then evaporated,yielding a drug-containing, lipid-saturated polymer matrix.

Exemplary Protocol I. Preparation of First Solution

Polymer (PLGA, PGA, PLA, or a combination thereof) and a polar lipidsuch as a sterol (e.g. cholesterol) and/or alpha- or gamma tocopheroland/or phosphatidyl ethanolamine are mixed into a volatile organicsolvent (e.g. ethyl acetate with/without chloroform). The mixture ismixed. The entire process is performed typically at room temperature. Afirst lipid-polymer mixture is thus obtained.

II. Preparation of Second Solution

The following materials are mixed with a volatile organic solvent(typically N-methylpyrrolidone [NMP], methanol, ethyl acetate orcombination thereof)

-   -   a. Active compound.    -   b. A phosphocholine or phosphatidylcholine derivative, e.g.        1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or        dioleoyl-phosphatidylcholine (DOPC), present as 30-90 mass % of        all lipids in the matrix, i.e. 30-90 mass % of phospholipids,        sterols, ceramides, fatty acids etc.    -   c. In some experiments a phosphatidylethanolamine e.g.        dimethyldimyristoyl phosphatidylethanolamine (DMPE) or        dipalmitoyl-phosphatidylethanolamine (DPPE)-present as 0.1-50        mass % of all lipids in the matrix.    -   d. In some experiments, a targeting moiety, e.g. a        fibronectin-hydrogenated phosphatidylethanolamine (HPE) complex,        is included as 0.1-10 mol % of all lipids in the matrix. To form        this complex, a fibronectin protein or fragment thereof        comprising the collagen-binding domain is bound to the amine        head group of HPE by a thioether bond.    -   e. In some experiments, 0.1-15 mass % of a free fatty acid, e.g.        linoleic acid (LN), or oleic acid (OA), is included as 0.1-10        mass % of all lipids in the matrix.    -   f. In some experiments, a salt, such as phosphate salts, is        included.        The second mixture is mixed, homogenized or sonicated. In some        cases, prior to mixing, homogenization or sonication, a        non-polar, volatile organic solvent, e.g. ethyl acetate, is        included with the mixture, which is stirred gently for 30        minutes. Typically the entire process is conducted at room        temperature, but higher temperatures of up to 45° C. are used,        typically when highly saturated lipids are used.        No water is required in the mixture.        III—Mixing the Polymer with the Drug/Protein Mixture

The second suspension (or solution) is added to the first solution understirring. Stirring is continued for up to 5 h. The entire process isperformed at room temperature and up to 60° C., all according to thespecific formulation, the nature of the lipids in use and the specificdrug. The resulting mixture should be homogenous.

IV—Evaporation of the Solvents

In some experiments, the solution from stage III is atomized into dry,heated air.

In other experiments, the solution from stage III is atomized intoethanol covered by liquid nitrogen or only liquid nitrogen withoutethanol, after which the nitrogen and/or ethanol (as above) areevaporated.

In other experiments, when coating of surfaces is performed; thesuspension from stage III is mixed with the particles or devices to becoated followed by evaporation of the volatile organic solvents. Theentire coating process is performed at a temperature of 30-60° C.

V—Vacuum Drying

Coated particles and coated devices are vacuum-dried for storage.

Example 2 Preparation of Doxycycline Hyclate-Bone Particles FillerFormulation for Treatment of Bone Infection (Osteomyelitis or Filling ofBone Effects Caused by Trauma) I. Preparation of First Solution

The following materials are mixed into ethyl acetate:

-   -   50-75 KDa PLGA (poly(lactic-co-glycolic acid, 85:15 ratio))    -   Cholesterol—50%-100% w/w vs. PLGA.

The mixture is mixed. The entire process is performed at roomtemperature. A fat-polymer combination matrix is thus obtained.

II. Preparation of Second Solution

The following materials are mixed with a volatile organic solvent(methanol and ethyl acetate)

-   -   a. Active compound,—an antibiotic Doxycycline hyclate    -   b. A        phosphatidylcholine,—1,2-distearoyl-sn-glycero-3-phosphocholine        (DSPC) present as 300-700% w/w vs. PLGA.        The mixture is mixed well. The entire process is conducted at        room temperature. When phosphatidylcholine having long saturated        fatty chains is used (e.g. DSPC) the process is typically        performed at higher temperatures of about 40-50° C.        No water is required in the mixture.        III—Mixing the Polymer with the Drug Mixture

The second solution or suspension is added to the first suspension,typically under stirring. Stirring is continued for 1-5 minutes. Theentire process is performed at a temperature of 20-50° C. depending ofthe lipid used.

IV—Evaporation Following Surface Coating

In order to coat bone filler particles, the particles are added to themixture of stage III followed by evaporation of the volatile organicsolvents. The entire process is performed at a temperature of 40-50° C.

The ratio between the volume and the percentage of solids in the mixtureof stage III and the mass of the bone particles will determine therelease period of the drug post hydration of the coated particles.

V—Vacuum Drying

Coated bone particles are vacuum-dried for storage.

The ability of bone filler particles (xenograftsbovine-commercial-BioOss®) coated with the matrix composition of thepresent invention to sustained release the drug (doxycycline hyclate) isshown in FIGS. 1 and 2. The rate of sustained release of Doxycyclinehyclate encapsulated within the matrix composition of the presentinvention was measured and compared to the release rate of the drug frombone particles socked with free DOX solution (having the same amount ofDOX). It was found that the drug was released constantly for about threeweeks (FIGS. 1A and B) following zero order kinetics (FIG. 2). Thevelocity values displayed in FIG. 2 were calculated following theassumption that the coated bone particles constitute only 20% of thetotal amount of bone particles used as bone fillers and the drug oncereleased, diffuses to the free volume between the bone particles.

The macro-structure of the bone particles before (FIG. 3A) and aftercoating with the matrix composition of the present invention (FIGS. 3Band 3C) was studied. As can be seen in FIG. 3, the structure of the boneparticles is not affected by coating; furthermore, the macro-structureof the bone particles is maintained even after 60 days of incubation inserum.

The structure of bone particles surfaces before (FIG. 4A) and aftercoating with the matrix composition of the present invention (FIG. 4B-E)was studied by SEM. The matrix composition contained DSPC, PLGA (85:15),cholesterol and the antibiotic doxycycline hyclate. The coated boneparticles were incubated in 10% FBS at 37° C. for 2 months. As can beseen the coating is homogenous and opaque, furthermore it covers most ofthe bone surface. With time, the coating is eliminated gradually, layerby layer by surface corrosion, during which the drug is being released.It can be further seen in FIG. 4E that after 60 days of incubation thecoating is almost completely eliminated, leaving the bone surface as inits original, uncoated form. The ordered structure of the matrixcomposition of the invention is shown in FIG. 5. Coated bone particles(PLGA (85:15), DPPC (16:0), and Cholesterol 10%), were analyzed byelectronic microscopy (X18,000) and negative staining (data not shown).The bright colored lines represent the polymer, where as the lipid isrepresented by the dark filling in between the polymeric material.

The influence of different polymer/lipid compositions on the releaserate of a given drug was measured using fluorescein entrapped within amatrix compositing PLGA (75:25), DPPC as the main phospholipid andvarying amounts of lauric acid (LA) and phosphatidylethanolamine (PE)having saturated fatty acid moieties of at least 14 carbons. As can beseen in FIG. 6 the expected release period of 90% of the entrappedmolecule was dramatically influenced by the LA:PE content. The releaselasted between about 20 to about 110 days when LA:PE w/w % ratio wasvaried between 10:10 to 0:0 respectively (vs. the total mass of theformulation). The drug release profile from bone particles coated withmatrix formulation comprising PE versus bone particles coated withmatrix formulation comprising cholesterol was compared. FIG. 7demonstrates that the release profiles of DOX from coated bone particlescomprising PE or cholesterol behaves similarly.

The fact that different polymer/lipid compositions influence the releaserate can be used to fulfill different clinical needs. For example, antiinflammation treatment with NSAID is commonly a short term treatment(e.g. few days) thus by using a fast degrading polymer such as PLGA50:50 and a 14:0 phospholipid such as DMPC, full release of the drug canbe completed within 10 days as can be seen in FIG. 8. In contrast, whenthe antibiotic drug, DOX, was associated with a slow degrading polymersuch as PLGA 85:15 and a 18:0 phospholipid such as DSPC, completerelease of the drug was accomplished after more than 50 days (FIG. 8).

The release profile of DOX from a mixture of coated and non coated bone(xenografts bovine—commercial—BioOss) particles was measured. The coatedparticles were mixed with similar plain (not-coated) bone particles in aratio of 1:4. As control we followed the release of a similar dose offree DOX from plain (not-coated) bone particles soaked with the freedrug. As can be seen in FIG. 9, following hydration (5% serum at 37° C.)the release of DOX from the formulation was not affected by the presenceof the non-coated particles. In comparison, most of the drug from theuncoated bone particles soaked with DOX was released shortly afterhydration (88% in 3 hours as compared to about 15% of the drug releasedfrom the coated bone particles during the same period). The formulationin this study comprised PLGA 75:25 and DPPC.

We have further demonstrated that the duration of drug release fromcoated bone particles is linearly depended on the formulation mass. Therelease of DOX from bone particles (12 mg/sample) coated with differentmass of the DOX containing matrix formulation was compared. Followinghydration (5% serum at 37° C.) the duration of release of 90% of theinitial DOX amount in the formulation was monitored (FIG. 10). Thelinear dependence between the duration of drug release and the mass ofthe coating matrix formulation suggests that the drug is released bygradual degradation of the matrix, and the release rate is not affectedby the overall mass of the formulation.

Example 3 Preparation of 1,3-Thiabendazole (TBZ) Formulation StockSolutions:

a. PLGA/ethyl acetate, 300 mg/ml (SS1, 1 ml): (i) Weight 300 mg PLGA(50:50; Sigma) into 4 ml glass vile, (ii) add 1 ml ethyl acetate, (iii)vortex for 5 minutes, (iv) Stir for 12-18 hours at room temperature(RT), (v) confirm that the polymer grains are totally dissolved, (vi)close under N₂, wrap with aluminum foil and keep it at RT, (vii) thesolution is good for 1 month.b. Cholesterol/ethyl acetate, 30 mg/ml (SS2, 1 ml): (i) Weight 30 mgcholesterol (Sigma 99%) into 4 ml glass vile, (ii) add 1 ml of ethylacetate, (iii) vortex for 5 minutes at RT, (iv) confirm that thecholesterol is totally dissolved. Otherwise, continue to vortex for more2 minutes, (v) close under N₂, wrap with aluminum foil and keep it atRT, (vi) the solution is good for 1 month.c. Ethyl acetate:Methanol 1:1 (SS2.1): (i) Put 10 ml of Ethyl acetateinto a 20 ml glass vile, (ii) add 10 ml methanol into the same vile,(iii) vortex for 20 seconds, (iv) keep the solution at RT, (v) theSolution is good for 1 month.d. Thiabendazole (TBZ)/ethyl acetate:methanol 1:1, 10 mg/ml (SS3, 1 ml):(i) weight 10 mg TBZ into 4 ml glass vile, (ii) add 1 ml of SS2.1 stocksolution, (iii) vortex for 5 minutes at RT, (iv) confirm that TBZ istotally dissolved. Otherwise, continue to vortex for more 2 minutes. Thesolution has some white turbidity, (v) close under N₂, wrap withaluminum foil and keep it at RT, (vi) the solution is good for 1 month.

Solution A (1.2 ml):

i. Add 1 ml SS2 (CH-EA, 30 mg CH) to 0.2 ml SS1 (PLGA/EA, 60 mg PLGA)into 4 ml glass vile.ii. Vortex for 5 minutes at RT.iii. Confirm that the mixture is uniform and lucid. Otherwise go back toii.iv. Close under N₂, wrap with aluminum foil and keep it at RT.v. The solution is good for 1 month.vi. Concentrations solution A: [CH]=25 mg/ml; [PLGA]=50 mg/ml.

Solution B (1 ml):

i. Weight 225 mg phospholipids (14:0) into 4 ml glass vile.ii. Add 0.75 ml SS3 (TBZ/EA-MET, 7.5 mg TBZ).iii. Add 0.25 ml ethyl acetate into the vile.iv. Vortex for 2 minutes at RT.v. Close under N₂, wrap with aluminum foil and keep it at RT.vi. The solution is good for 1 month.vii. Concentrations: [phospholipid (14:0)]=225 mg/ml, [TBZ]=7.5 mg/ml.

Solution C (1 ml):

i. Pour 0.4 ml of solution B into a 4 ml glass vile.ii. Add 0.6 ml of solution A into the vile.iii. Vortex for 2 minutes at RT.iv. Check: The solution is liquid at RT, has a pale yellow color withsome turbidity.v. Close under N₂ and wrap with aluminum foil.vi. Concentrations: [CH]=15 mg/ml; [PLGA]=30 mg/ml; [14:0]=90 mg/ml;[TBZ]=3 mg/ml.

Bone Coated Preparation:

i. Weight 12.5 (±0.5) mg bone particles (Bio-Oss or EndoBon) into 1.8 mlglass vile;ii. Wash the bones with purified water (½ ml DDW); pump out the waterwith the micropipette, followed by vacuum for 12-18 hours.iii. Prepare a heating block, heated to 45° C.iv. Heat solution C to 45° C. for 30 seconds. Make sure that thesolution totally melts and becomes uniform.v. Add 50 μl of solution C to the bone particles with a 10-100 μlmicropipette.vi. Put the 1.8 ml vials, unsealed, in the heating block (45° C.) for 30minutes.vii. Remove from heating and close with a stopper.viii. vacuum the (half-sealed) vials with a rotation pump (1×10-1 Torr)for 12-18 hours.ix. Separate gently the fused bone particles with a spatula.x. Transfer the dry coated bone particles into a new 4 ml glass vile;xi. Close under N₂, wrap with aluminum foil and keep it at RT.xii. The coated bone particles are good for 1 month.The release profile of TBZ from bone particles coated with the TBZcontaining matrix composition after hydration (5% serum, 37° C.) can beseen in FIG. 11.

Example 4 Drug Release from Absorbable Gelatin Sponge Containing theSustained Release Formulation of the Invention

A solution containing PLGA—75:25, PC 16:0, cholesterol 10% andDoxycycline hyclate (DOX) 10% was injected into the center of theabsorbable gelatin sponge foam cube (Gelatamp. ROEKO). The overallcontent of DOX in the injected formulation was 380 μg in 25 μl. Thesolvent was evaporated in a 37° C. incubator and subsequently over nightunder vacuum. As a control, the common use of pre-wetting with a similardose of DOX (380 μg) solution was injected into the gelatin sponge tube.Following hydration (5% serum at 37° C.) the release of DOX from thegelatin sponge cube into the surrounding was detected and quantified byHPLC. As can be seen in FIG. 12, while in the control sample, the totalamount of DOX was released immediately into the medium, only about 40%of the DOX associated with the PLGA/PC/cholesterol formulation wasreleased into the medium immediately after hydration, while the rest ofthe drug was gradually released for more than 7 days.

Example 5

SEM elements analysis of coated bone particles: Bone particles coatedwith a matrix formulation (PLGA 50:50, cholesterol and DPPC 16:0) andnon coated bone particles were analyzed by SEM element analysis. Theelement analysis of bone particle surface, coated and non-coated aresummarized in the tables 1 and 2 below:

TABLE 1 non coated bone particle surface. Element Wt % At % CK 10.0516.91 NK 02.27 03.28 OK 43.97 55.55 NaK 00.67 00.59 MgK 00.76 00.63 PK11.59 07.57 CaK 30.68 15.47 Matrix Correction ZAF

TABLE 2 coated bone particle surface. Element Wt % At % CK 42.28 55.73NK 02.96 03.34 OK 30.90 30.57 NaK 00.46 00.32 MgK 00.39 00.25 PK 5.9403.04 CaK 17.08 06.75 Matrix Correction ZAF

The element analysis shows that carbon (CK in tables 1 and 2) is thedominant element in bone particles coated with the formulation of theinvention. Carbon is the major element in both the polymer used in theformulation (PLGA 50:50) and the lipid used (DPPC 16:0). In contrast,the contents of calcium and phosphate which are dominant elements of theplain bone particles (uncoated bone particles) are at least two timeslower on the surface of the coated bone particles.

The gradual degradation of the bone particle coating formulation of thepresent invention after hydration was studied by SEM element analysis.The weight percentages of carbon, calcium and phosphate atoms on thesurface of the coated bone particles were monitored by SEM. As can beseen in FIG. 13, following hydration of the coated bone particles thepercentage of the carbon atoms on the surface of the coated boneparticles decreases with time, whereas the percentages of calcium andphosphate increase with time. These results demonstrate that upongradual degradation of the coating formulation the surface of the boneparticles is gradually exposed.

Example 6 Elevated Turbidity in the Supernatant of Bone Particles Coatedwith Formulations of the Invention is Correlated with the Appearance ofVesicles in the Supernatant

Bone particles (TCP artificial bone substitute—commercial) coated with aformulation of the present invention comprising doxycycline hyclate—DOX)were hydrated in 5% serum at 37° C. After 1 hour the bone particles wereseparated from the supernatant and the supernatant was analyzed bymonitoring its absorbance at 520 nm. The bone particles werere-incubated in a fresh 5% of serum at 37° C. for another 23 hours.After 23 hours the bone particles were separated from the supernatantand the later was analyzed as described before.

Results: The supernatant collected from plain uncoated bone particlesdid not have significant turbidity. In contrast, after 1 h of hydrationa significant turbidity was evidenced in supernatants of coated boneparticles. Three types of bone particles coatings were tested: (i) theformulation of the present invention comprising an antibiotic agent and(ii) DPPC and an antibiotic agent coating and (iii) PLGA coating. Boneparticles that were coated with PLGA showed smaller increase inturbidity (OD₅₂₀ nm˜0.85) as compared to the DPPC coated particles(OD₅₂₀ nm>3) or the particles coated with the formulation of theinvention (OD₅₂₀ nm˜2.0) (FIG. 14A). After additional 23 hours ofincubation (hydration) under the same conditions the turbidity was muchlower than that measured after 1 h, and evidenced only in bone coatingformulations containing lipids (formulations (i) and (ii)) (FIG. 14B).

The supernatant removed from plain non coated bone particles as well ascoated bone particles ((i), (ii) and (iii) as described above) after thesecond round of incubation for 23 hours, were further analyzed byelectron microscopy (magnitude of 18,000) and negative staining. In thesupernatants taken from bone particles coated with the formulation ofthe present invention (i) or with DPPC (ii), vesicle like structures ofdifferent sizes were evidenced (FIG. 14C).

The nature of the released material from bone particles coated with amatrix formulation containing PLGA (85:15), DPPC (16:0) and theantibiotic drug (DOX), was further analyzed by a Size Distributor(Malvern Instruments DST ver.5). The coated bone particles were hydratedwith 5% serum and incubated for 24 hour at 37° C. The supernatant wasremoved after 24 hours and analyzed. The released material wascharacterized by two particle populations having an average size of550.3 nm and 4.2 nm (FIG. 14D). The zeta potential of the particles,measured using the same instrument, was found to be close to zero(0.0225 mV) (FIG. 14E). The diameter size of the released material aswell as its neutral charge may suggest that these vesicle like particlesare composed mainly from the DPPC found in the coating matrixformulation.

The described turbidity experiments suggest that the post hydrationturbidity seen in the supernatants of hydrated coated bone particles ispredominantly controlled by the lipid content of the formulation. Theinitial high turbidity which is follows by a slower elevation of theturbidity is correlated with the kinetic behavior of DOX release frombone particles coated with the formulation of the invention (FIG. 2) aswell as the kinetic behavior of NBD-marked fluorescent phospholipidsrelease (FIG. 15) which is characterized by an initial burst releasefollowed by slow zero order release.

Example 7 Small Angle X-Ray Scattering Analysis of Bone Particles Coatedwith the Matrix Composition of the Invention

We have analyzed the structure of bone particles (TCP artificial bonesubstitute—commercial) coated with a matrix composition comprising thebiopolymer PLGA 85:15, the lipid—DPPC 16:0 and the antibioticdrug—doxycycline hyclate—DOX. The dried particles were loaded into aglass capillary and analyzed by Small Angle X-ray scattering.

Results: The scattering profile of bone particles coated with the matrixformulation as described above, suggest that the matrix formulation hasan ordered structure with several sub structures of various sizesranging from 5 nm to 40 nm (FIG. 16). The structure of a driedphospholipids powder was further analyzed and was found to have anordered structure having sub-structures smaller than 5 nm. As a control,the structure of plain, uncoated, TCP particles was studied and it wasfound that it is not characterized by the presence of sub structures ofless than 1 nm. Thus, the sub structures observed in the scatteringprofile of coated bone particles can be attributed to the coatingmaterial itself and not to the plain uncoated bone particles.

Example 8 Differential Scanning Calorimetric Analysis (DSC) of PolymerContaining Solution (Solution A) with or without Cholesterol

Vacuum dried polymer (PLGA (75:25)) was analyzed by differentialscanning calorimeter. The temperature of the polymer was raised at arate of 5° C./min with or without cholesterol (Cstrl) in a differentPolymer/cholesterol mass ratio (w/w). A typical calorimetric reaction ofPLGA (with no Cstrl) displays an intake of heat during heating up to200° C. due to PLGA melting. In contrast, the addition of cholesteroldecreases the heat uptake by the polymer in a dose response manner, upto the level where almost no heat uptake is evidenced. The narrow heatuptake at about 150° C. is typical the free cholesterol in this system(FIG. 17A). The effect of cholesterol was not affected by the rate ofheating (data not shown). Similar but lower effect was evidenced whenother lipids such as Alfa tocopherol were introduced to the polymer. Incontrast, the heat uptake by the polymer upon heating was unaffected bythe presence of fatty acids such as mineral oil (carbonic chainC12-C18). (FIG. 17B)

Example 9

Coating metal implants by the matrix formulations of the invention.Dental implant made of titanium was coated by a matrix formulation (PLGA85:15, DSPC 18:0, cholesterol 10% and DOX 10%) by immersing the metal inthe final solution containing the matrix composition. (see step IIIexample 1) The solvent was than evaporated in an incubator at 37° C.,followed by continuous drying under vacuum over night (FIG. 18)

Example 10 Pre-Clinical Testing of Matrix Composition of the PresentInvention for Bone Recovery Animal Models:

A. Tibial osteomyelitis in RabbitB. Bacteria: staphylococcus aureusAll preclinical testing is performed in accordance with the guidelinesfor Regulation of Animal Experiments in the State of Israel andaccording to the Ethics Committee of the research institution.Test A): Determine the relevant bacterial load for the model:1. Cause a trauma to the bone (as determined in test A)—10 animals.2. Fill the void (injured bone) by tricalcium phosphate (TCP) materialand seal it with Bone-Wax.3. Load the site with defined amount of bacteria by injecting it intothe site.4. Duration—˜22 days. Clinical signs and body weight (3× weekly) ismonitored.5. At the end of the incubation time: bleed the animal for basicHematology & Biochemistry blood (prior to the termination of the test).6. X-Ray of the tibia prior to the termination of the test (day ˜20)7. terminate the experiment, and harvest the tibia for bacteriologicaltest.8. extract the bacteria from the bone and determine the bacterialconcentration (as described below)

Determination of bacterial concentration in the bone marrow: The bonemarrow and the intramedullary canal is swabbed with sterile cotton tipapplicators for gross culture analysis of quality assurance. Theinoculated applicator is streaked onto blood plates and then placed into5 mL of sterile TSB. The plates and tubes are then incubated at 37° C.for 24 h and growth is recorded.

Determination of bacterial concentration in the per gram of bone: Thebone is placed into a sterile, 50 mL centrifuge tube and weighed. Thebone is then crushed and the final product weighed. Normal sterilesaline, 0.9%, is added in a 3:1 ratio (3 mL saline/g of bone), and thesuspensions are vortexed for 2 min. Six 10-fold dilutions of eachsuspension are prepared with sterile, normal saline, 0.9%. Samples (20μL) of each dilution, including the initial suspension, are plated, intriplicate, onto blood agar plates and incubated at 37° C. for 24 h;colony forming units are counted at the greatest dilution for each tibiasample. The S. aureus concentration is calculated in CFU/g of bone.

Test A) Determine the Relevant Bacterial Load for the Model:

Addition No of Treat- Dura- Group Trauma of Bacteria animals ment tion ATest Positive Yes (L) 3 TCP 22 days (control) B Test Positive Yes (M) 3TCP 22 days (control) C Test Positive Yes (H) 3 TCP 22 days (control) DCon- Negative No 1 TCP 22 days trol (control)

Test B) Determine the Bactericidal Activity of the Matrix Composition ofthe Invention:

1. Cause a trauma to the bone (as described in test A)—13 animals2. Fill the void (injured bone) by TCP material and seal it withBone-Wax.3. Loading the site with defined amount of bacteria by injecting it intothe site (the load will be determine following the result of test A).4. Duration—˜22 days. Clinical signs and body weight (3× weekly) ismonitored.5. During the incubation time: bleed the animals for basic Hematology &Biochemistry blood panel at day 7 and 16 (prior to the termination ofthe test).6. X-Ray of the tibia at day 1 (or 2)+at day ˜20 prior to thetermination of the test.7. Terminate the experiment, and to harvest the tibia forbacteriological tests.8. Extracting the bacteria from the bone and determining the bacterialconcentration: as described above for test A.9. Local drug concentration is assayed.

Test B) Determine the Bactericidal Activity of the Matrix Composition ofthe Invention (BonyPid):

Addition No of Treat- Dura- Group Trauma of Bacteria animals ment tion ATest Positive Yes 6 BonyPid 22 days B Test Positive Yes 6 TCP 22 days(control) C Con- Positive no 1 TCP 22 days trol (control)

Test C) Toxicology of the Matrix Composition of the Invention:

1. Cause a trauma to the bone (as described in test A)—24 animals2. Fill the void (injured bone) by TCP material and seal it withBone-Wax.3. Loading the site with defined amount of bacteria by injecting it intothe site (the load will be determine following the result of test A).4. Duration—˜45 days. Clinical signs and body weight (3× weekly) aremonitored. Termination time is determined according to the X-Ray resultstaken during the incubation time.5. During the incubation time: bleed the animals for basic Hematology &Biochemistry blood panel at day 0, 10, 30 and 45 (prior to thetermination of the test).6. The animals will be bleeding for blood-drug-concentration analysis atdays 1, 3, 10, 16 and 30.7. X-Ray of the tibia at day 2, 20, 30 and 43 prior to the terminationof the test.8. Terminate the experiment and harvest the tibia for Histology tests.9. Histology tests for the injured site to 50% of the animals (12animals).10. Extracting the bacteria from the bone and determining the bacterialconcentration for 50% of the animals (12 animals) as described above.

Test C) Toxicology of the Matrix Composition of the Invention (BonyPid):

Addition No of Treat- Dura- Group Trauma of Bacteria animals ment tion ATest Positive Yes 6 BonyPid 45 days C Test Positive Yes 6 BonyPid 45days D Con- Positive no 6 BonyPid 45 days trol F Con- Positive no 6BonyPid 45 days trol

Example 12 Pre-Clinical Testing in a Periodontitis Animal Model

In a three-stage study, experimental periodontitis in induced in pigsusing a cotton ligature placed in a submarginal position. Theperiodontitis is treated by a combination of scaling and root planing(SRP) and the one of the following treatments:

-   -   Local application of a matrix implant containing very high,        high, medium, or low doses (30, 15, 5, and 1 mg/application        site, respectively; represented as VH, H, M, and L,        respectively) of flurbiprofen and doxycycline.    -   Local application of a matrix implant containing no active        ingredient, at amounts of matrix corresponding to the amounts of        matrix included with the high, medium, and low doses described        above (negative control).    -   Local application of flurbiprofen and doxycycline, at doses        corresponding to the very high, high, medium, and low doses        described above, administered as free drug.    -   Systemic twice-daily administration of flurbiprofen and        doxycycline, at doses corresponding to the high, medium, and low        doses described above. This (in combination with SRP) is        considered the reference standard for treatment of periodontitis        in this animal model.    -   No treatment (additional negative control group).        The following parameters are measured daily in each group:    -   Carrier marker levels, in order to determine in vivo stability        of the carrier in the tissue.    -   Levels of flurbiprofen, doxycycline, and their known metabolites        in the site of application, the surrounding tissue, and the        circulation.    -   Toxicity tests.    -   In addition, the following indicia of efficacy are determined:        -   Improvement in clinical parameters such as probing depth            (PD), clinical attachment level (CAL), and bleeding on            probing (BOP).        -   Improvement in radiological parameters such as the distance            between the cemento-enamel junction and the alveolar bone            crest.        -   Histologic analysis.

The number of pigs in each group, study length, and groups in each stageof the study are set forth in Tables 1-3 below:

STAGE 1 Experimental groups Dose Number Days Control — 2 42 Free drugsVH 2 42 Free drugs H 2 42 Matrix- no drug VH 2 42 Matrix- no drug H 2 42Matrix with drug VH 2 42 Matrix with drug H 2 42

STAGE 2 Experimental groups Dose Number Days Control — 2 42 Free drugs M2 42 Free drugs L 2 42 Matrix- no drug M 2 42 Matrix- no drug L 2 42Matrix with drug M 2 42 Matrix with drug L 2 42

STAGE 3 Experimental groups Dose Number Days Control — 2 56 Referencestandard H 3 56 Reference standard M 3 56 Reference standard L 3 56 Freedrugs H 3 56 Free drugs M 3 56 Free drugs L 3 56 Matrix- no drug H 3 56Matrix- no drug M 3 56 Matrix- no drug L 3 56 Matrix with drug H 3 56Matrix with drug M 3 56 Matrix with drug L 3 56

Example 13 Clinical Testing of Matrix Compositions of the PresentInvention for Periodontitis

The following study tests the safety and clinical, radiological, andmicrobiologic effects of matrix compositions of the present inventionwhen used as an adjunct to scaling and root planing (SRP).

Scaling and root planing is the most common and conservative form oftreatment for periodontal (gum) disease. Scaling is the removal ofcalculus and plaque that attach to the tooth surfaces. The processespecially targets the area below the gum line, along the root. Plaqueis more likely to stick to rough surfaces. For this reason, the rootsurface is smoothed down in a process called root planing. Root planingremoves any remaining calculus and smoothes irregular areas of the rootsurface

Study design is longitudinal, randomized, single-masked, andinter-subject. Male and female subjects, aged 20-65 with moderate tosevere chronic periodontitis or aggressive periodontitis, are recruited.Detailed medical and dental histories are obtained. Exclusioncriteria: 1) a complicating systemic condition, i.e. pregnancy ordiabetes; 2) use of systemic antibiotic or NSAID drugs in the past 3months; 3) smoking; 4) any known allergy to ingredients of the matrixcomposition; 5) periodontal treatment undertaken less than 6 monthsprior to baseline. Subjects undergo SRP either alone or in combinationwith administration of (a) matrix implants containing antibiotic+NSAIDdrugs; (b) matrix implants containing no active ingredient; (c) orallyadministered free antibiotic+NSAID drugs; or (d) systemicantibiotic+NSAID drugs. Implants or free antibiotics are administered atmultiple sites in the oral cavity.

The following clinical measurements are recorded at baseline, and at 1,3, 6, and 9 months: probing depth (PD), clinical attachment level (CAL),bleeding on probing (BOP), and gingivitis, plaque, and staining indices.

Microbiological tests, including bacterial culturing andN-benzoyl-DL-arginine-napthylamide (BANA) tests, are performed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

1-48. (canceled)
 49. A matrix composition comprising: a. a biodegradablepolymer in association with a first lipid having a polar group; b. asecond lipid comprising at least one phospholipid having hydrocarbonchains of at least 14 carbons; c. at least one pharmaceutical activeagent; wherein the matrix composition is essentially devoid of water andis adapted for providing sustained release of the pharmaceutical activeagent.
 50. The matrix composition of claim 49, wherein said phospholipidis a phosphatidylcholine having fatty acid moieties having at least 14carbons.
 51. The matrix composition of claim 49 wherein thebiodegradable polymer is a biodegradable polyester selected from thegroup consisting of PLA (polylactic acid), PGA (poly glycolic acid) andPLGA (Poly (lactic co glycolic acid).
 52. The matrix composition ofclaim 49 wherein the pharmaceutical active agent is selected from anantibiotic, an antifungal, a non-steroidal anti-inflammatory drug, asteroid, an anti-cancer agent, an osteogenic factor and a boneresorption inhibitor.
 53. The matrix composition of claim 49, whereinthe first lipid having a polar group comprises a sterol.
 54. The matrixcomposition of claim 49, wherein the weight ratio of total lipids tosaid biodegradable polymer is between 1.5:1 and 9:1 inclusive.
 55. Thematrix composition of claim 49, wherein said matrix composition ishomogeneous.
 56. The matrix composition of claim 49, further comprisinga tocopherol; a compound selected from the group consisting ofsphingolipid; a free fatty acid having 14 or more carbon atom; anadditional phospholipid selected from the group consisting of aphosphatidylserine, a phosphatidylglycerol, and a phosphatidylinositol;and a targeting moiety capable of interacting with a target moleculeselected from the group consisting of a collagen molecule, a fibrinmolecule and a heparin.
 57. The matrix composition of claim 53, whereinsaid sterol is a cholesterol.
 58. The matrix composition of claim 57,wherein said cholesterol is present in an amount of 5-50 mole percent ofthe total lipid content of said matrix composition.
 59. The matrixcomposition of claim 49 for the sustained release of said pharmaceuticalactive agent, wherein at least 50% of said pharmaceutical active agentis released from the composition at zero-order kinetics.
 60. An implantcomprising the matrix composition of claim
 49. 61. A pharmaceuticalcomposition for sustained release of an active agent comprising thematrix composition of claim
 49. 62. The pharmaceutical composition ofclaim 61 where the active agent is selected from an antibiotic, anantifungal, a non-steroidal anti-inflammatory drug, a steroid, andanti-cancer agent, an osteogenic factor and a bone resorption inhibitor.63. A method of administering a pharmaceutically active agent selectedfrom the group consisting of an antibiotic, an antifungal, anon-steroidal anti-inflammatory drug, a steroid, an anti-cancer agent,an osteogenic factor and a bone resorption inhibitor to a subject inneed thereof, said method comprising the step of administering to saidsubject the matrix composition of claim 52, thereby administering thepharmaceutically active agent to the subject.
 64. A method of treatingperiodontitis in a subject in need thereof, said method comprising thestep of administering to said subject the matrix composition of claim52, thereby treating periodontitis in the subject.
 65. A method ofstimulating bone augmentation in a subject in need thereof, said methodcomprising the step of administering to said subject the matrixcomposition of claim 52, wherein the pharmaceutically active agent isselected from an osteogenic factor and a bone resorption inhibitor,thereby stimulating bone augmentation in a subject in need thereof. 66.A medical device comprising: a substrate and a biocompatible coatingdeposited on at least a fraction of said substrate, wherein saidbiocompatible coating comprises the matrix composition of claim
 49. 67.The medical device of claim 66, wherein said biocompatible coatingincludes multi-layers.
 68. The medical device of claim 66, wherein saidsubstrate is selected from orthopedic nails, orthopedic screws,orthopedic staples, orthopedic wires, orthopedic pins, metal orpolymeric implants, bone filler particles, collagen and non-collagenmembranes, suture materials, orthopedic cements and sponges.
 69. Use ofthe composition of claim 49 for the coating of substrates selected fromorthopedic nails, orthopedic screws, orthopedic staples, orthopedicwires, orthopedic pins, metal or polymeric implants, bone fillerparticles, collagen and non-collagen membranes, suture materials,orthopedic cements and sponges.
 70. A method of producing a matrixcomposition, said method comprising the steps of: a. mixing into a firstvolatile organic solvent: (i) a biodegradable polymer and (ii) a firstlipid having a polar group; b. mixing into a second volatile organicsolvent: (i) at least one pharmaceutical active agent; (ii) a secondlipid selected from phospholipids having hydrocarbon chains of at least14 carbons; and c. mixing the products resulting from steps (a) and (b),to produce a homogeneous mixture; and d. evaporating the volatileorganic solvents; wherein each step of said method is substantially freeof an aqueous solution, thereby producing a homogeneous matrixcomposition.
 71. The method of claim 70, wherein said phospholipid is aphosphatidylcholine having fatty acid moieties having at least 14carbons.
 72. The method of claim 70, wherein said first lipid is aphosphatidylethanolamine having fatty acid moieties having at least 14carbons.
 73. The method of claim 70, wherein said biodegradable polymeris a polyester selected from the group consisting of PLA (polylacticacid), PGA (poly glycolic acid) and PLGA (poly(lactic-co-glycolicacid)).
 74. The method of claim 70, wherein said first lipid is asterol.